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The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione <t>Sepharose</t> and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014
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1) Product Images from "A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly"

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

Journal: eLife

doi: 10.7554/eLife.03473

The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014
Figure Legend Snippet: The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014

Techniques Used: Mutagenesis, Binding Assay, Transformation Assay, Western Blot, Fluorescence In Situ Hybridization, Labeling, Staining, Recombinant, Incubation, SDS Page, Expressing, Fluorescence, Microscopy

Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011
Figure Legend Snippet: Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011

Techniques Used: Sequencing, Incubation, SDS Page, Staining, Western Blot, Stable Transfection, In Vitro

RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012
Figure Legend Snippet: RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012

Techniques Used: Incubation, SDS Page, Staining, Western Blot

eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005
Figure Legend Snippet: eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005

Techniques Used: Mutagenesis, Western Blot, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Staining, Sedimentation, Centrifugation, Agarose Gel Electrophoresis, Northern Blot

Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007
Figure Legend Snippet: Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007

Techniques Used: Purification, Isolation, Agarose Gel Electrophoresis, Northern Blot, Silver Staining, Western Blot

GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010
Figure Legend Snippet: GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010

Techniques Used: Sedimentation, Western Blot, Recombinant, Incubation, SDS Page, Staining, Transformation Assay, Binding Assay, Expressing, Fluorescence, Microscopy, Mutagenesis

eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009
Figure Legend Snippet: eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009

Techniques Used: Recombinant, Incubation, Purification, SDS Page, Staining, Western Blot

2) Product Images from "A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly"

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

Journal: eLife

doi: 10.7554/eLife.03473

The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014
Figure Legend Snippet: The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014

Techniques Used: Mutagenesis, Binding Assay, Transformation Assay, Western Blot, Fluorescence In Situ Hybridization, Labeling, Staining, Recombinant, Incubation, SDS Page, Expressing, Fluorescence, Microscopy

Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011
Figure Legend Snippet: Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011

Techniques Used: Sequencing, Incubation, SDS Page, Staining, Western Blot, Stable Transfection, In Vitro

RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012
Figure Legend Snippet: RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012

Techniques Used: Incubation, SDS Page, Staining, Western Blot

eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005
Figure Legend Snippet: eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005

Techniques Used: Mutagenesis, Western Blot, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Staining, Sedimentation, Centrifugation, Agarose Gel Electrophoresis, Northern Blot

Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007
Figure Legend Snippet: Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007

Techniques Used: Purification, Isolation, Agarose Gel Electrophoresis, Northern Blot, Silver Staining, Western Blot

GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010
Figure Legend Snippet: GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010

Techniques Used: Sedimentation, Western Blot, Recombinant, Incubation, SDS Page, Staining, Transformation Assay, Binding Assay, Expressing, Fluorescence, Microscopy, Mutagenesis

eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009
Figure Legend Snippet: eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009

Techniques Used: Recombinant, Incubation, Purification, SDS Page, Staining, Western Blot

3) Product Images from "Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿"

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00708-06

The RCC1 propeller increases substrate affinity for importins α3 and α4. Competition binding assays were performed using substrates containing the same NLS fused to different protein core domains. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with two competing substrates in equimolar amounts and importin β. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used in the competition experiments: (A) RCC1 and NLS(R)-core, (B) NLS(N)-prop and NLS(N)-core, and (C) NLS(N)-prop and nucleoplasmin. α, importin α; β, importin β; NPL, nucleoplasmin.
Figure Legend Snippet: The RCC1 propeller increases substrate affinity for importins α3 and α4. Competition binding assays were performed using substrates containing the same NLS fused to different protein core domains. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with two competing substrates in equimolar amounts and importin β. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used in the competition experiments: (A) RCC1 and NLS(R)-core, (B) NLS(N)-prop and NLS(N)-core, and (C) NLS(N)-prop and nucleoplasmin. α, importin α; β, importin β; NPL, nucleoplasmin.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining

Importin α binding specificity of RCC1 and nucleoplasmin is represented in in vitro binding assays. (A) Coomassie staining of bead-associated proteins derived from binding reactions of RCC1 with the various α importins. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with purified recombinant RCC1. Bound proteins were separated by SDS-PAGE. The asterisk marks bovine serum albumin that was used in the binding reactions at a concentration of 2 mg/ml and remains to some extent in the beads despite the washing procedure. (B) Binding assays of RCC1 and RCC1Δ24 with immobilized importin α3-GST in the presence of importin β. Bound and unbound fractions were subjected to SDS-PAGE and analyzed by Coomassie staining (bound proteins) or Western blotting using an RCC1-specific antibody (unbound proteins and input samples). + or −, presence or absence of RCC1 and RCC1Δ24, respectively. (C) Binding of nucleoplasmin to the various α importins, as described for panel A. (D) Similar to panel A, with the exception that binding reactions were carried out with the nucleoplasmin core domain in the presence of importin β. Bound proteins and an input sample were separated by SDS-PAGE and analyzed by Coomassie staining. α, importin α; β, importin β; NPL, nucleoplasmin.
Figure Legend Snippet: Importin α binding specificity of RCC1 and nucleoplasmin is represented in in vitro binding assays. (A) Coomassie staining of bead-associated proteins derived from binding reactions of RCC1 with the various α importins. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with purified recombinant RCC1. Bound proteins were separated by SDS-PAGE. The asterisk marks bovine serum albumin that was used in the binding reactions at a concentration of 2 mg/ml and remains to some extent in the beads despite the washing procedure. (B) Binding assays of RCC1 and RCC1Δ24 with immobilized importin α3-GST in the presence of importin β. Bound and unbound fractions were subjected to SDS-PAGE and analyzed by Coomassie staining (bound proteins) or Western blotting using an RCC1-specific antibody (unbound proteins and input samples). + or −, presence or absence of RCC1 and RCC1Δ24, respectively. (C) Binding of nucleoplasmin to the various α importins, as described for panel A. (D) Similar to panel A, with the exception that binding reactions were carried out with the nucleoplasmin core domain in the presence of importin β. Bound proteins and an input sample were separated by SDS-PAGE and analyzed by Coomassie staining. α, importin α; β, importin β; NPL, nucleoplasmin.

Techniques Used: Binding Assay, In Vitro, Staining, Derivative Assay, Incubation, Purification, Recombinant, SDS Page, Concentration Assay, Western Blot

Both basic clusters of the RCC1 N terminus contribute to importin α binding. Equal amounts of the indicated C-terminally GST-tagged importins were used in binding experiments with single substrates. The importin-GSTs were immobilized on glutathione-Sepharose beads and incubated with substrates in the presence (A) or in both the presence (+) and absence (B, C, and D) of importin β. Bound proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used: (A) RCC1, (B) RCC1mt2 (K8A, R9A), (C) RCC1Δ13, and (D) RCC1mt3 (K21A, K22A). α, importin α; β, importin β; Imp., importin.
Figure Legend Snippet: Both basic clusters of the RCC1 N terminus contribute to importin α binding. Equal amounts of the indicated C-terminally GST-tagged importins were used in binding experiments with single substrates. The importin-GSTs were immobilized on glutathione-Sepharose beads and incubated with substrates in the presence (A) or in both the presence (+) and absence (B, C, and D) of importin β. Bound proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used: (A) RCC1, (B) RCC1mt2 (K8A, R9A), (C) RCC1Δ13, and (D) RCC1mt3 (K21A, K22A). α, importin α; β, importin β; Imp., importin.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining

Importin α binding specificity of the second basic cluster of the RCC1 N terminus is mediated by the protein context. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with NLS(RΔ12)-core either in the absence or in the presence (+) of importin β. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. α, importin α; β, importin β; Imp., importin.
Figure Legend Snippet: Importin α binding specificity of the second basic cluster of the RCC1 N terminus is mediated by the protein context. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with NLS(RΔ12)-core either in the absence or in the presence (+) of importin β. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. α, importin α; β, importin β; Imp., importin.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining

) is underlined. Basic clusters involved in importin α binding are in bold. The NLS-containing nucleoplasmin region was cloned N terminal to the core domain to achieve NLS(N)-core. NLS(R)-core was generated by fusing the RCC1 N terminus listed in panel A N terminal to the core domain. Amino acids 13 to 35 of RCC1 were cloned N terminal to the core domain to obtain NLS(RΔ12)-core. (C) Coomassie staining of bead-associated importins. Equal amounts of the indicated importins were immobilized via their C-terminal GST tag on glutathione-Sepharose beads and separated by SDS-PAGE. Mass spectrometry analysis of tryptic cleavage peptides confirmed the integrity of the N termini and revealed that contaminations of the importin α4-GST preparation (enclosed by the circle) are GST fragments. α, importin α; β, importin β.
Figure Legend Snippet: ) is underlined. Basic clusters involved in importin α binding are in bold. The NLS-containing nucleoplasmin region was cloned N terminal to the core domain to achieve NLS(N)-core. NLS(R)-core was generated by fusing the RCC1 N terminus listed in panel A N terminal to the core domain. Amino acids 13 to 35 of RCC1 were cloned N terminal to the core domain to obtain NLS(RΔ12)-core. (C) Coomassie staining of bead-associated importins. Equal amounts of the indicated importins were immobilized via their C-terminal GST tag on glutathione-Sepharose beads and separated by SDS-PAGE. Mass spectrometry analysis of tryptic cleavage peptides confirmed the integrity of the N termini and revealed that contaminations of the importin α4-GST preparation (enclosed by the circle) are GST fragments. α, importin α; β, importin β.

Techniques Used: Binding Assay, Clone Assay, Generated, Staining, SDS Page, Mass Spectrometry

The NLS is not sufficient to mediate importin α binding specificity. Equal amounts of the indicated C-terminally GST-tagged importins were used in binding experiments with single substrates. The importin-GSTs were immobilized on glutathione-Sepharose beads and incubated with the purified recombinant His-tagged proteins. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. Binding experiments were carried out with the following substrates: (A) NLS(N)-core, (B) NLS(N)-prop, and (C) NLS(R)-core. α, importin α; β, importin β.
Figure Legend Snippet: The NLS is not sufficient to mediate importin α binding specificity. Equal amounts of the indicated C-terminally GST-tagged importins were used in binding experiments with single substrates. The importin-GSTs were immobilized on glutathione-Sepharose beads and incubated with the purified recombinant His-tagged proteins. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. Binding experiments were carried out with the following substrates: (A) NLS(N)-core, (B) NLS(N)-prop, and (C) NLS(R)-core. α, importin α; β, importin β.

Techniques Used: Binding Assay, Incubation, Purification, Recombinant, SDS Page, Staining

The NLS contributes to importin α binding specificity. Competition binding assays were performed using substrates differing only in their NLS. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with one single substrate (A) or with two competing substrates in equimolar amounts in the presence of importin β (B and C). Bound proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used: (A) zzRCC1, (B) zzRCC1 and NLS(N)-prop, and (C) NLS(R)-core and NLS(N)-core. α, importin α; β, importin β.
Figure Legend Snippet: The NLS contributes to importin α binding specificity. Competition binding assays were performed using substrates differing only in their NLS. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with one single substrate (A) or with two competing substrates in equimolar amounts in the presence of importin β (B and C). Bound proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used: (A) zzRCC1, (B) zzRCC1 and NLS(N)-prop, and (C) NLS(R)-core and NLS(N)-core. α, importin α; β, importin β.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining

4) Product Images from "Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿"

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00708-06

The RCC1 propeller increases substrate affinity for importins α3 and α4. Competition binding assays were performed using substrates containing the same NLS fused to different protein core domains. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with two competing substrates in equimolar amounts and importin β. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used in the competition experiments: (A) RCC1 and NLS(R)-core, (B) NLS(N)-prop and NLS(N)-core, and (C) NLS(N)-prop and nucleoplasmin. α, importin α; β, importin β; NPL, nucleoplasmin.
Figure Legend Snippet: The RCC1 propeller increases substrate affinity for importins α3 and α4. Competition binding assays were performed using substrates containing the same NLS fused to different protein core domains. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with two competing substrates in equimolar amounts and importin β. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used in the competition experiments: (A) RCC1 and NLS(R)-core, (B) NLS(N)-prop and NLS(N)-core, and (C) NLS(N)-prop and nucleoplasmin. α, importin α; β, importin β; NPL, nucleoplasmin.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining

Importin α binding specificity of RCC1 and nucleoplasmin is represented in in vitro binding assays. (A) Coomassie staining of bead-associated proteins derived from binding reactions of RCC1 with the various α importins. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with purified recombinant RCC1. Bound proteins were separated by SDS-PAGE. The asterisk marks bovine serum albumin that was used in the binding reactions at a concentration of 2 mg/ml and remains to some extent in the beads despite the washing procedure. (B) Binding assays of RCC1 and RCC1Δ24 with immobilized importin α3-GST in the presence of importin β. Bound and unbound fractions were subjected to SDS-PAGE and analyzed by Coomassie staining (bound proteins) or Western blotting using an RCC1-specific antibody (unbound proteins and input samples). + or −, presence or absence of RCC1 and RCC1Δ24, respectively. (C) Binding of nucleoplasmin to the various α importins, as described for panel A. (D) Similar to panel A, with the exception that binding reactions were carried out with the nucleoplasmin core domain in the presence of importin β. Bound proteins and an input sample were separated by SDS-PAGE and analyzed by Coomassie staining. α, importin α; β, importin β; NPL, nucleoplasmin.
Figure Legend Snippet: Importin α binding specificity of RCC1 and nucleoplasmin is represented in in vitro binding assays. (A) Coomassie staining of bead-associated proteins derived from binding reactions of RCC1 with the various α importins. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with purified recombinant RCC1. Bound proteins were separated by SDS-PAGE. The asterisk marks bovine serum albumin that was used in the binding reactions at a concentration of 2 mg/ml and remains to some extent in the beads despite the washing procedure. (B) Binding assays of RCC1 and RCC1Δ24 with immobilized importin α3-GST in the presence of importin β. Bound and unbound fractions were subjected to SDS-PAGE and analyzed by Coomassie staining (bound proteins) or Western blotting using an RCC1-specific antibody (unbound proteins and input samples). + or −, presence or absence of RCC1 and RCC1Δ24, respectively. (C) Binding of nucleoplasmin to the various α importins, as described for panel A. (D) Similar to panel A, with the exception that binding reactions were carried out with the nucleoplasmin core domain in the presence of importin β. Bound proteins and an input sample were separated by SDS-PAGE and analyzed by Coomassie staining. α, importin α; β, importin β; NPL, nucleoplasmin.

Techniques Used: Binding Assay, In Vitro, Staining, Derivative Assay, Incubation, Purification, Recombinant, SDS Page, Concentration Assay, Western Blot

Both basic clusters of the RCC1 N terminus contribute to importin α binding. Equal amounts of the indicated C-terminally GST-tagged importins were used in binding experiments with single substrates. The importin-GSTs were immobilized on glutathione-Sepharose beads and incubated with substrates in the presence (A) or in both the presence (+) and absence (B, C, and D) of importin β. Bound proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used: (A) RCC1, (B) RCC1mt2 (K8A, R9A), (C) RCC1Δ13, and (D) RCC1mt3 (K21A, K22A). α, importin α; β, importin β; Imp., importin.
Figure Legend Snippet: Both basic clusters of the RCC1 N terminus contribute to importin α binding. Equal amounts of the indicated C-terminally GST-tagged importins were used in binding experiments with single substrates. The importin-GSTs were immobilized on glutathione-Sepharose beads and incubated with substrates in the presence (A) or in both the presence (+) and absence (B, C, and D) of importin β. Bound proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used: (A) RCC1, (B) RCC1mt2 (K8A, R9A), (C) RCC1Δ13, and (D) RCC1mt3 (K21A, K22A). α, importin α; β, importin β; Imp., importin.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining

Importin α binding specificity of the second basic cluster of the RCC1 N terminus is mediated by the protein context. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with NLS(RΔ12)-core either in the absence or in the presence (+) of importin β. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. α, importin α; β, importin β; Imp., importin.
Figure Legend Snippet: Importin α binding specificity of the second basic cluster of the RCC1 N terminus is mediated by the protein context. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with NLS(RΔ12)-core either in the absence or in the presence (+) of importin β. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. α, importin α; β, importin β; Imp., importin.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining

) is underlined. Basic clusters involved in importin α binding are in bold. The NLS-containing nucleoplasmin region was cloned N terminal to the core domain to achieve NLS(N)-core. NLS(R)-core was generated by fusing the RCC1 N terminus listed in panel A N terminal to the core domain. Amino acids 13 to 35 of RCC1 were cloned N terminal to the core domain to obtain NLS(RΔ12)-core. (C) Coomassie staining of bead-associated importins. Equal amounts of the indicated importins were immobilized via their C-terminal GST tag on glutathione-Sepharose beads and separated by SDS-PAGE. Mass spectrometry analysis of tryptic cleavage peptides confirmed the integrity of the N termini and revealed that contaminations of the importin α4-GST preparation (enclosed by the circle) are GST fragments. α, importin α; β, importin β.
Figure Legend Snippet: ) is underlined. Basic clusters involved in importin α binding are in bold. The NLS-containing nucleoplasmin region was cloned N terminal to the core domain to achieve NLS(N)-core. NLS(R)-core was generated by fusing the RCC1 N terminus listed in panel A N terminal to the core domain. Amino acids 13 to 35 of RCC1 were cloned N terminal to the core domain to obtain NLS(RΔ12)-core. (C) Coomassie staining of bead-associated importins. Equal amounts of the indicated importins were immobilized via their C-terminal GST tag on glutathione-Sepharose beads and separated by SDS-PAGE. Mass spectrometry analysis of tryptic cleavage peptides confirmed the integrity of the N termini and revealed that contaminations of the importin α4-GST preparation (enclosed by the circle) are GST fragments. α, importin α; β, importin β.

Techniques Used: Binding Assay, Clone Assay, Generated, Staining, SDS Page, Mass Spectrometry

The NLS is not sufficient to mediate importin α binding specificity. Equal amounts of the indicated C-terminally GST-tagged importins were used in binding experiments with single substrates. The importin-GSTs were immobilized on glutathione-Sepharose beads and incubated with the purified recombinant His-tagged proteins. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. Binding experiments were carried out with the following substrates: (A) NLS(N)-core, (B) NLS(N)-prop, and (C) NLS(R)-core. α, importin α; β, importin β.
Figure Legend Snippet: The NLS is not sufficient to mediate importin α binding specificity. Equal amounts of the indicated C-terminally GST-tagged importins were used in binding experiments with single substrates. The importin-GSTs were immobilized on glutathione-Sepharose beads and incubated with the purified recombinant His-tagged proteins. Bead-associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. Binding experiments were carried out with the following substrates: (A) NLS(N)-core, (B) NLS(N)-prop, and (C) NLS(R)-core. α, importin α; β, importin β.

Techniques Used: Binding Assay, Incubation, Purification, Recombinant, SDS Page, Staining

The NLS contributes to importin α binding specificity. Competition binding assays were performed using substrates differing only in their NLS. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with one single substrate (A) or with two competing substrates in equimolar amounts in the presence of importin β (B and C). Bound proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used: (A) zzRCC1, (B) zzRCC1 and NLS(N)-prop, and (C) NLS(R)-core and NLS(N)-core. α, importin α; β, importin β.
Figure Legend Snippet: The NLS contributes to importin α binding specificity. Competition binding assays were performed using substrates differing only in their NLS. Equal amounts of the indicated C-terminally GST-tagged importins were immobilized on glutathione-Sepharose beads and incubated with one single substrate (A) or with two competing substrates in equimolar amounts in the presence of importin β (B and C). Bound proteins were separated by SDS-PAGE and visualized by Coomassie staining. The following substrates were used: (A) zzRCC1, (B) zzRCC1 and NLS(N)-prop, and (C) NLS(R)-core and NLS(N)-core. α, importin α; β, importin β.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining

5) Product Images from "The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein"

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm614

Rrp47p is a multimeric complex. ( A ) Recombinant His( 6 )-Rrp47p purification. Samples were resolved through a 15% SDS–PAGE gel and visualized with Coomassie blue G250. Lane 1, non-induced cell extract; lane 2, cell extract after 4 h induction; lane 3, Ni-NTA superflow eluate; lane 4, SP-sepharose non-bound fraction, lane 5, SP-sepharose eluate; lane 6, peak fraction from the superdex 200 GF column. The positions of molecular weight markers (in kDa) are indicated on the left. ( B ) Gel filtration analysis of the SP-sepharose retained fraction. The A 280 profile is shown, together with the elution volumes of the markers thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.3 kDa). The calibration curve obtained from the molecular weight markers is shown.
Figure Legend Snippet: Rrp47p is a multimeric complex. ( A ) Recombinant His( 6 )-Rrp47p purification. Samples were resolved through a 15% SDS–PAGE gel and visualized with Coomassie blue G250. Lane 1, non-induced cell extract; lane 2, cell extract after 4 h induction; lane 3, Ni-NTA superflow eluate; lane 4, SP-sepharose non-bound fraction, lane 5, SP-sepharose eluate; lane 6, peak fraction from the superdex 200 GF column. The positions of molecular weight markers (in kDa) are indicated on the left. ( B ) Gel filtration analysis of the SP-sepharose retained fraction. The A 280 profile is shown, together with the elution volumes of the markers thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.3 kDa). The calibration curve obtained from the molecular weight markers is shown.

Techniques Used: Recombinant, Purification, SDS Page, Molecular Weight, Filtration

Residues C-terminal to the PMC2NT domain contribute to Rrp47p interaction. ( A ) Schematic of the N-terminal region of Rrp6p. The location of the PMC2NT domain and the four short α-helices α1–α4 are shown. Residues P176 and L197 define the C-termini of truncated Rrp6p constructs. These residues are mutated to stop codons and therefore not the C-terminal residues. ( B ) Pull-down assay of His( 6 )-Rrp47p with GST-tagged N-terminal Rrp6p fragments. Eluates from glutathione-sepharose beads were resolved by SDS–PAGE and assayed by western blot analyses with primary antisera against GST (upper panel) or the His tag (lower panel). Electrophoretic mobilites of protein molecular weight markers (in kDa) are given on the left.
Figure Legend Snippet: Residues C-terminal to the PMC2NT domain contribute to Rrp47p interaction. ( A ) Schematic of the N-terminal region of Rrp6p. The location of the PMC2NT domain and the four short α-helices α1–α4 are shown. Residues P176 and L197 define the C-termini of truncated Rrp6p constructs. These residues are mutated to stop codons and therefore not the C-terminal residues. ( B ) Pull-down assay of His( 6 )-Rrp47p with GST-tagged N-terminal Rrp6p fragments. Eluates from glutathione-sepharose beads were resolved by SDS–PAGE and assayed by western blot analyses with primary antisera against GST (upper panel) or the His tag (lower panel). Electrophoretic mobilites of protein molecular weight markers (in kDa) are given on the left.

Techniques Used: Construct, Pull Down Assay, SDS Page, Western Blot, Molecular Weight

Rrp47p interacts with nucleic acid when bound to Rrp6p. Glutathione-sepharose beads charged with GST or a GST fusion protein containing the N-terminal region of Rrp6p (GST-Rrp6Δ212-721) were incubated with lysate containing His( 6 )-Rrp47p or a control extract (pRSETB). After washing the beads, protein complexes were incubated with radiolabelled E. coli tRNA Phe or a DNA restriction fragment. The non-bound material was removed and bound tRNA or DNA was determined by quantifying the associated Cherenkov radiation. ( A ) Levels of tRNA retained. ( B ) Levels of DNA retained. The results shown are the average of three independent assays, the error bars indicating the associated range.
Figure Legend Snippet: Rrp47p interacts with nucleic acid when bound to Rrp6p. Glutathione-sepharose beads charged with GST or a GST fusion protein containing the N-terminal region of Rrp6p (GST-Rrp6Δ212-721) were incubated with lysate containing His( 6 )-Rrp47p or a control extract (pRSETB). After washing the beads, protein complexes were incubated with radiolabelled E. coli tRNA Phe or a DNA restriction fragment. The non-bound material was removed and bound tRNA or DNA was determined by quantifying the associated Cherenkov radiation. ( A ) Levels of tRNA retained. ( B ) Levels of DNA retained. The results shown are the average of three independent assays, the error bars indicating the associated range.

Techniques Used: Incubation

Rrp47p binds directly to the N-terminal region of Rrp6p. ( A ) Rrp47p co-purifies with Rrp6p in pull-down assays. Glutathione-sepharose beads were incubated first with lysate containing recombinant GST–Rrp6p or GST and then with lysate from cells expressing His( 6 )-Rrp47p or containing the control vector pRSETB. Eluates were resolved through a 10% SDS–PAGE gel and analysed by western blotting using an antibody specific for the His tag. O denotes the top of the resolving gel; X marks the position of the bromophenol blue tracking dye. ( B ) Schematic of the domain structure of Rrp6p. PMC2NT, exonuclease (EXO) and HRDC domains are indicated as boxes. Deletions within Rrp6p constructs made in this study are indicated by broken lines. ( C and D ) Western analyses of eluates from pull-down assays performed using different Rrp6p constructs, as in A. Upper panels show western blots of eluates using an anti-GST antibody; lower panels show corresponding results with an anti-His antibody. Multiple bands are visible with the anti-GST antibody due to proteolytic degradation of Rrp6p. The electrophoretic migration of the full-length Rrp6p protein constructs are indicated to the right of the panels.
Figure Legend Snippet: Rrp47p binds directly to the N-terminal region of Rrp6p. ( A ) Rrp47p co-purifies with Rrp6p in pull-down assays. Glutathione-sepharose beads were incubated first with lysate containing recombinant GST–Rrp6p or GST and then with lysate from cells expressing His( 6 )-Rrp47p or containing the control vector pRSETB. Eluates were resolved through a 10% SDS–PAGE gel and analysed by western blotting using an antibody specific for the His tag. O denotes the top of the resolving gel; X marks the position of the bromophenol blue tracking dye. ( B ) Schematic of the domain structure of Rrp6p. PMC2NT, exonuclease (EXO) and HRDC domains are indicated as boxes. Deletions within Rrp6p constructs made in this study are indicated by broken lines. ( C and D ) Western analyses of eluates from pull-down assays performed using different Rrp6p constructs, as in A. Upper panels show western blots of eluates using an anti-GST antibody; lower panels show corresponding results with an anti-His antibody. Multiple bands are visible with the anti-GST antibody due to proteolytic degradation of Rrp6p. The electrophoretic migration of the full-length Rrp6p protein constructs are indicated to the right of the panels.

Techniques Used: Incubation, Recombinant, Expressing, Plasmid Preparation, SDS Page, Western Blot, Construct, Migration

Rrp47p interacts with the N-terminal region of Rrp6p in yeast. ( A–D ) Western analyses of cell extracts from strains expressing epitope-tagged forms of Rrp47p and/or Rrp6p. Extracts were resolved through 10% SDS–PAGE gels, protein transferred to western blots and the fusion proteins detected using PAP or GST-specific antisera. Relevant strain genotypes are indicated above each lane. The rrp47-zz, rrp6-TAP and zz-rrp6 alleles encode full-length fusion proteins, whereas the zz-Rrp6Δ1-213 and the GST-Rrp6Δ212-721 proteins are truncated Rrp6p variants. The GST fusion proteins were expressed under the control of the GAL promoter; panel D shows analyses of extracts from strains grown in glucose-based media (glc) and in galactose-based media (gal). The upper panel in D shows a western analysis using the GST-specific antibody. The centre panel in D shows a western analysis of the same samples using the PAP antibody. Bands corresponding to the detected fusion proteins are indicated on the right. SDS–PAGE analyses (lower panels) indicate the relative loading of each extract. ( E ) Western analysis of lysates from isogenic wild-type and rrp47- Δ strains, using a His( 6 )-Rrp47p antiserum. Proteins were resolved through a 15% SDS–PAGE gel. The migration of size markers (in kDa) is indicated on the left. A strong band of the expected size is detected in the wild-type lysate (lane 1) and absent in the rrp47-Δ extract (lane 2). ( F ) Western analyses of the bound fractions of extracts from strains expressing GST (lane 1) or GST-Rrp6Δ212-721 (lane 2) after incubation with glutathione-sepharose beads. The upper panel shows a blot probed with the GST-specific antibody, the lower panel shows a blot probed with the His( 6 )-Rrp47p antiserum.
Figure Legend Snippet: Rrp47p interacts with the N-terminal region of Rrp6p in yeast. ( A–D ) Western analyses of cell extracts from strains expressing epitope-tagged forms of Rrp47p and/or Rrp6p. Extracts were resolved through 10% SDS–PAGE gels, protein transferred to western blots and the fusion proteins detected using PAP or GST-specific antisera. Relevant strain genotypes are indicated above each lane. The rrp47-zz, rrp6-TAP and zz-rrp6 alleles encode full-length fusion proteins, whereas the zz-Rrp6Δ1-213 and the GST-Rrp6Δ212-721 proteins are truncated Rrp6p variants. The GST fusion proteins were expressed under the control of the GAL promoter; panel D shows analyses of extracts from strains grown in glucose-based media (glc) and in galactose-based media (gal). The upper panel in D shows a western analysis using the GST-specific antibody. The centre panel in D shows a western analysis of the same samples using the PAP antibody. Bands corresponding to the detected fusion proteins are indicated on the right. SDS–PAGE analyses (lower panels) indicate the relative loading of each extract. ( E ) Western analysis of lysates from isogenic wild-type and rrp47- Δ strains, using a His( 6 )-Rrp47p antiserum. Proteins were resolved through a 15% SDS–PAGE gel. The migration of size markers (in kDa) is indicated on the left. A strong band of the expected size is detected in the wild-type lysate (lane 1) and absent in the rrp47-Δ extract (lane 2). ( F ) Western analyses of the bound fractions of extracts from strains expressing GST (lane 1) or GST-Rrp6Δ212-721 (lane 2) after incubation with glutathione-sepharose beads. The upper panel shows a blot probed with the GST-specific antibody, the lower panel shows a blot probed with the His( 6 )-Rrp47p antiserum.

Techniques Used: Western Blot, Expressing, SDS Page, Gas Chromatography, Migration, Incubation

6) Product Images from "Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2"

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2

Journal: Nature Communications

doi: 10.1038/s41467-018-06160-x

DBA-linked Tsr2E64G mutant is impaired in binding ESS2. a hTsr2E64G mutant expressed in yeast impairs yeast growth. Overexpression of eS26 rescues from the impaired growth. The conditional P GAL1-TSR2 strain was co-transformed with 2μ vectors expressing indicated genes. Transformants were spotted in 10-fold dilutions on repressive glucose containing media and grown at indicated temperatures for 2–4 days. b Tsr2 DBA mutant cells (hTsr2E64G) accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - TSR2 cells were grown at 30 °C in glucose containing media to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. c hTsr2E64G mutant inefficiently binds eS26. GST-hTsr2 and GST-hTsr2E64G were immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant eS26 and incubated at indicated salt concentrations. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE, visualized by Coomassie Blue staining. L = input (1:10 diluted). The error bars show the standard deviation. d The far-UV CD spectra of both human WT Tsr2 and the E64G-DBA variant. The midpoints of the thermal denaturation curves followed at 222 nm are at 57 °C for both proteins. e Isothermal titration calorimetry (ITC) measurements of human Tsr2 with human ESS2. The binding isotherms were plotted against the molar ratio. The measured parameters and K d values are indicated within the plots
Figure Legend Snippet: DBA-linked Tsr2E64G mutant is impaired in binding ESS2. a hTsr2E64G mutant expressed in yeast impairs yeast growth. Overexpression of eS26 rescues from the impaired growth. The conditional P GAL1-TSR2 strain was co-transformed with 2μ vectors expressing indicated genes. Transformants were spotted in 10-fold dilutions on repressive glucose containing media and grown at indicated temperatures for 2–4 days. b Tsr2 DBA mutant cells (hTsr2E64G) accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - TSR2 cells were grown at 30 °C in glucose containing media to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. c hTsr2E64G mutant inefficiently binds eS26. GST-hTsr2 and GST-hTsr2E64G were immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant eS26 and incubated at indicated salt concentrations. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE, visualized by Coomassie Blue staining. L = input (1:10 diluted). The error bars show the standard deviation. d The far-UV CD spectra of both human WT Tsr2 and the E64G-DBA variant. The midpoints of the thermal denaturation curves followed at 222 nm are at 57 °C for both proteins. e Isothermal titration calorimetry (ITC) measurements of human Tsr2 with human ESS2. The binding isotherms were plotted against the molar ratio. The measured parameters and K d values are indicated within the plots

Techniques Used: Mutagenesis, Binding Assay, Over Expression, Transformation Assay, Expressing, Fluorescence In Situ Hybridization, Labeling, Staining, Incubation, Recombinant, SDS Page, Standard Deviation, Variant Assay, Isothermal Titration Calorimetry

C-terminal acidic tail of Tsr2 keeps eS26-RNA free. a eS26 co-enriches nucleic acids. b RNase A triggers aggregation of eS26. GST-eS26 was treated with RNase A and incubated for 10 min at RT in a photometric cuvette. c Tsr2 prevents aggregation of recombinant eS26 in vitro. Thirty-three micromolar GST-eS26 and a two-fold concentration of Tsr2 (66 µM) in PBSKMT was pre-incubated for 1 h at 4 °C (final volume: 90 µl). One microgram of RNase A was added to initiate aggregation. After 1 h of incubation, the scattering signal of the aggregated eS26 was monitored at 450 nm ( Y -axes). Three replicates for each well were measured. The error bars show the standard deviation. d Tsr2 releases RNA bound to GST-eS26. RNA was extracted from immobilized GST-eS26 after addition of increasing amounts of Tsr2 or Tsr2-N, respectively, separated on a 1% agarose gel and stained by EtBr. e GST-Tsr2-C was immobilized on Glutathione Sepharose before incubation with purified Tsr2, Tsr2-N or/and an E. coli lysate containing recombinant eS26 in the presence or absence of RNaseA. L = input (1:10 diluted)
Figure Legend Snippet: C-terminal acidic tail of Tsr2 keeps eS26-RNA free. a eS26 co-enriches nucleic acids. b RNase A triggers aggregation of eS26. GST-eS26 was treated with RNase A and incubated for 10 min at RT in a photometric cuvette. c Tsr2 prevents aggregation of recombinant eS26 in vitro. Thirty-three micromolar GST-eS26 and a two-fold concentration of Tsr2 (66 µM) in PBSKMT was pre-incubated for 1 h at 4 °C (final volume: 90 µl). One microgram of RNase A was added to initiate aggregation. After 1 h of incubation, the scattering signal of the aggregated eS26 was monitored at 450 nm ( Y -axes). Three replicates for each well were measured. The error bars show the standard deviation. d Tsr2 releases RNA bound to GST-eS26. RNA was extracted from immobilized GST-eS26 after addition of increasing amounts of Tsr2 or Tsr2-N, respectively, separated on a 1% agarose gel and stained by EtBr. e GST-Tsr2-C was immobilized on Glutathione Sepharose before incubation with purified Tsr2, Tsr2-N or/and an E. coli lysate containing recombinant eS26 in the presence or absence of RNaseA. L = input (1:10 diluted)

Techniques Used: Incubation, Recombinant, In Vitro, Concentration Assay, Standard Deviation, Agarose Gel Electrophoresis, Staining, Purification

Eukaryotic-specific segments of eS26 are required to bind Tsr2. a XL-MS reveals crosslinks between ESS2 and N-terminal domain of Tsr2. The crosslinked residues are listed in the Supplementary Table 1 . b Phylogenetic analyses for eS26 and Tsr2. ESS1, ESS2 from eS26 and Tsr2 are present only in eukaryotes. c Sequence alignment of yeast S26 compared to the indicated species. 70 d ESSs in eS26 are required to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant WT eS26, eS26 deficient in ESS1 and/or ESS2 or archaeal eS26 from Sulfolobus solfataricus . Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining. L = input (1:10 diluted). e Residues 99–109 in eS26-ESS2 are necessary to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with an E. coli lysate containing recombinant WT eS26 or eS26 with variant truncations in C-terminal ESS2. Samples were analyzed as in d . Results from in vitro binding were quantified using ImageJ. f ESS1 and ESS2 deletion from eS26 causes slow growth phenotype in yeast. The conditional P GAL1 - RPS26Arps26b∆ strain was transformed with WT or the indicated truncations of eS26 and spotted in 10-fold dilutions on repressive glucose containing media and grown at 25 °C for 4 days. g Cells with eS26 lacking ESS1 or ESS2 accumulate immature 20S pre-rRNA in the cytoplasm. Localization of 20S pre-rRNA in P GAL1 - RPS26Arps26b∆ cells transformed with indicated plasmids was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. h Overexpression of ProtA-FLAG-eS26 is toxic in yeast. The WT yeast strain (BY4741) was transformed with ProtA-FLAG-eS26 or ProtA-FLAG-eS26 lacking ESS2, spotted in 10-fold dilutions on galactose containing media and grown at 25 °C for 4 days. i FLAG-ESS2 fusion protein co-precipitates Tsr2. ESS2 was purified using ProteinA-Tev-FLAG tag, the FLAG eluate was TCA precipitated, separated by SDS-PAGE, and analyzed by Coomassie staining and western analyses using the indicated antibodies
Figure Legend Snippet: Eukaryotic-specific segments of eS26 are required to bind Tsr2. a XL-MS reveals crosslinks between ESS2 and N-terminal domain of Tsr2. The crosslinked residues are listed in the Supplementary Table 1 . b Phylogenetic analyses for eS26 and Tsr2. ESS1, ESS2 from eS26 and Tsr2 are present only in eukaryotes. c Sequence alignment of yeast S26 compared to the indicated species. 70 d ESSs in eS26 are required to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant WT eS26, eS26 deficient in ESS1 and/or ESS2 or archaeal eS26 from Sulfolobus solfataricus . Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining. L = input (1:10 diluted). e Residues 99–109 in eS26-ESS2 are necessary to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with an E. coli lysate containing recombinant WT eS26 or eS26 with variant truncations in C-terminal ESS2. Samples were analyzed as in d . Results from in vitro binding were quantified using ImageJ. f ESS1 and ESS2 deletion from eS26 causes slow growth phenotype in yeast. The conditional P GAL1 - RPS26Arps26b∆ strain was transformed with WT or the indicated truncations of eS26 and spotted in 10-fold dilutions on repressive glucose containing media and grown at 25 °C for 4 days. g Cells with eS26 lacking ESS1 or ESS2 accumulate immature 20S pre-rRNA in the cytoplasm. Localization of 20S pre-rRNA in P GAL1 - RPS26Arps26b∆ cells transformed with indicated plasmids was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. h Overexpression of ProtA-FLAG-eS26 is toxic in yeast. The WT yeast strain (BY4741) was transformed with ProtA-FLAG-eS26 or ProtA-FLAG-eS26 lacking ESS2, spotted in 10-fold dilutions on galactose containing media and grown at 25 °C for 4 days. i FLAG-ESS2 fusion protein co-precipitates Tsr2. ESS2 was purified using ProteinA-Tev-FLAG tag, the FLAG eluate was TCA precipitated, separated by SDS-PAGE, and analyzed by Coomassie staining and western analyses using the indicated antibodies

Techniques Used: Mass Spectrometry, Sequencing, In Vitro, Incubation, Recombinant, SDS Page, Staining, Variant Assay, Binding Assay, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Over Expression, Purification, FLAG-tag, Western Blot

ESSs mediate Tsr2-dependent importin:eS26 complex disassembly. a Tsr2 cannot efficiently dissociate the Kap123:eS26ΔESS1ΔESS2 FLAG complex. The complex of GST-Kap123 with eS26 FLAG , eS26ΔESS1 FLAG eS26ΔESS2 FLAG or eS26ΔESS1ΔESS2 FLAG was immobilized on Glutathione Sepharose and incubated with either buffer alone or with Tsr2 before pull-down. L = input (1:10 diluted). b Tsr2 DWI mutant does not dissociate a Kap123:eS26 complex. The complex of GST-Kap123 with eS26 FLAG was incubated with either buffer alone, purified Tsr2 or Tsr2 DWI before pull-down. L = input (1:10 diluted). c Efficient recruitment of eS26 to pre-40S requires both of the ESSs. Enp1-TAP was isolated from P GAL1 - RPS26Arps26b∆ strain transformed with WT eS26 or eS26 lacking ESS1 or ESS2. After tandem affinity purification, eluates were separated by 4–12% gradient SDS-PAGE and subjected to western analyses using indicated antibodies. Protein levels of uS7, uS3 served as a loading control. d Protein levels of eS26, eS26ΔESS1, eS26ΔESS2 and eS26ΔESS1ΔESS2 in whole-cell extracts of P GAL1 - RPS26Arps26b∆ strain were determined by western analyses using α-eS26 antibodies. Gsp1 protein levels served as a loading control
Figure Legend Snippet: ESSs mediate Tsr2-dependent importin:eS26 complex disassembly. a Tsr2 cannot efficiently dissociate the Kap123:eS26ΔESS1ΔESS2 FLAG complex. The complex of GST-Kap123 with eS26 FLAG , eS26ΔESS1 FLAG eS26ΔESS2 FLAG or eS26ΔESS1ΔESS2 FLAG was immobilized on Glutathione Sepharose and incubated with either buffer alone or with Tsr2 before pull-down. L = input (1:10 diluted). b Tsr2 DWI mutant does not dissociate a Kap123:eS26 complex. The complex of GST-Kap123 with eS26 FLAG was incubated with either buffer alone, purified Tsr2 or Tsr2 DWI before pull-down. L = input (1:10 diluted). c Efficient recruitment of eS26 to pre-40S requires both of the ESSs. Enp1-TAP was isolated from P GAL1 - RPS26Arps26b∆ strain transformed with WT eS26 or eS26 lacking ESS1 or ESS2. After tandem affinity purification, eluates were separated by 4–12% gradient SDS-PAGE and subjected to western analyses using indicated antibodies. Protein levels of uS7, uS3 served as a loading control. d Protein levels of eS26, eS26ΔESS1, eS26ΔESS2 and eS26ΔESS1ΔESS2 in whole-cell extracts of P GAL1 - RPS26Arps26b∆ strain were determined by western analyses using α-eS26 antibodies. Gsp1 protein levels served as a loading control

Techniques Used: Incubation, Mutagenesis, Purification, Isolation, Transformation Assay, Affinity Purification, SDS Page, Western Blot

7) Product Images from "Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2"

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2

Journal: Nature Communications

doi: 10.1038/s41467-018-06160-x

DBA-linked Tsr2E64G mutant is impaired in binding ESS2. a hTsr2E64G mutant expressed in yeast impairs yeast growth. Overexpression of eS26 rescues from the impaired growth. The conditional P GAL1-TSR2 strain was co-transformed with 2μ vectors expressing indicated genes. Transformants were spotted in 10-fold dilutions on repressive glucose containing media and grown at indicated temperatures for 2–4 days. b Tsr2 DBA mutant cells (hTsr2E64G) accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - TSR2 cells were grown at 30 °C in glucose containing media to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. c hTsr2E64G mutant inefficiently binds eS26. GST-hTsr2 and GST-hTsr2E64G were immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant eS26 and incubated at indicated salt concentrations. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE, visualized by Coomassie Blue staining. L = input (1:10 diluted). The error bars show the standard deviation. d The far-UV CD spectra of both human WT Tsr2 and the E64G-DBA variant. The midpoints of the thermal denaturation curves followed at 222 nm are at 57 °C for both proteins. e Isothermal titration calorimetry (ITC) measurements of human Tsr2 with human ESS2. The binding isotherms were plotted against the molar ratio. The measured parameters and K d values are indicated within the plots
Figure Legend Snippet: DBA-linked Tsr2E64G mutant is impaired in binding ESS2. a hTsr2E64G mutant expressed in yeast impairs yeast growth. Overexpression of eS26 rescues from the impaired growth. The conditional P GAL1-TSR2 strain was co-transformed with 2μ vectors expressing indicated genes. Transformants were spotted in 10-fold dilutions on repressive glucose containing media and grown at indicated temperatures for 2–4 days. b Tsr2 DBA mutant cells (hTsr2E64G) accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - TSR2 cells were grown at 30 °C in glucose containing media to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. c hTsr2E64G mutant inefficiently binds eS26. GST-hTsr2 and GST-hTsr2E64G were immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant eS26 and incubated at indicated salt concentrations. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE, visualized by Coomassie Blue staining. L = input (1:10 diluted). The error bars show the standard deviation. d The far-UV CD spectra of both human WT Tsr2 and the E64G-DBA variant. The midpoints of the thermal denaturation curves followed at 222 nm are at 57 °C for both proteins. e Isothermal titration calorimetry (ITC) measurements of human Tsr2 with human ESS2. The binding isotherms were plotted against the molar ratio. The measured parameters and K d values are indicated within the plots

Techniques Used: Mutagenesis, Binding Assay, Over Expression, Transformation Assay, Expressing, Fluorescence In Situ Hybridization, Labeling, Staining, Incubation, Recombinant, SDS Page, Standard Deviation, Variant Assay, Isothermal Titration Calorimetry

C-terminal acidic tail of Tsr2 keeps eS26-RNA free. a eS26 co-enriches nucleic acids. b RNase A triggers aggregation of eS26. GST-eS26 was treated with RNase A and incubated for 10 min at RT in a photometric cuvette. c Tsr2 prevents aggregation of recombinant eS26 in vitro. Thirty-three micromolar GST-eS26 and a two-fold concentration of Tsr2 (66 µM) in PBSKMT was pre-incubated for 1 h at 4 °C (final volume: 90 µl). One microgram of RNase A was added to initiate aggregation. After 1 h of incubation, the scattering signal of the aggregated eS26 was monitored at 450 nm ( Y -axes). Three replicates for each well were measured. The error bars show the standard deviation. d Tsr2 releases RNA bound to GST-eS26. RNA was extracted from immobilized GST-eS26 after addition of increasing amounts of Tsr2 or Tsr2-N, respectively, separated on a 1% agarose gel and stained by EtBr. e GST-Tsr2-C was immobilized on Glutathione Sepharose before incubation with purified Tsr2, Tsr2-N or/and an E. coli lysate containing recombinant eS26 in the presence or absence of RNaseA. L = input (1:10 diluted)
Figure Legend Snippet: C-terminal acidic tail of Tsr2 keeps eS26-RNA free. a eS26 co-enriches nucleic acids. b RNase A triggers aggregation of eS26. GST-eS26 was treated with RNase A and incubated for 10 min at RT in a photometric cuvette. c Tsr2 prevents aggregation of recombinant eS26 in vitro. Thirty-three micromolar GST-eS26 and a two-fold concentration of Tsr2 (66 µM) in PBSKMT was pre-incubated for 1 h at 4 °C (final volume: 90 µl). One microgram of RNase A was added to initiate aggregation. After 1 h of incubation, the scattering signal of the aggregated eS26 was monitored at 450 nm ( Y -axes). Three replicates for each well were measured. The error bars show the standard deviation. d Tsr2 releases RNA bound to GST-eS26. RNA was extracted from immobilized GST-eS26 after addition of increasing amounts of Tsr2 or Tsr2-N, respectively, separated on a 1% agarose gel and stained by EtBr. e GST-Tsr2-C was immobilized on Glutathione Sepharose before incubation with purified Tsr2, Tsr2-N or/and an E. coli lysate containing recombinant eS26 in the presence or absence of RNaseA. L = input (1:10 diluted)

Techniques Used: Incubation, Recombinant, In Vitro, Concentration Assay, Standard Deviation, Agarose Gel Electrophoresis, Staining, Purification

Eukaryotic-specific segments of eS26 are required to bind Tsr2. a XL-MS reveals crosslinks between ESS2 and N-terminal domain of Tsr2. The crosslinked residues are listed in the Supplementary Table 1 . b Phylogenetic analyses for eS26 and Tsr2. ESS1, ESS2 from eS26 and Tsr2 are present only in eukaryotes. c Sequence alignment of yeast S26 compared to the indicated species. 70 d ESSs in eS26 are required to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant WT eS26, eS26 deficient in ESS1 and/or ESS2 or archaeal eS26 from Sulfolobus solfataricus . Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining. L = input (1:10 diluted). e Residues 99–109 in eS26-ESS2 are necessary to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with an E. coli lysate containing recombinant WT eS26 or eS26 with variant truncations in C-terminal ESS2. Samples were analyzed as in d . Results from in vitro binding were quantified using ImageJ. f ESS1 and ESS2 deletion from eS26 causes slow growth phenotype in yeast. The conditional P GAL1 - RPS26Arps26b∆ strain was transformed with WT or the indicated truncations of eS26 and spotted in 10-fold dilutions on repressive glucose containing media and grown at 25 °C for 4 days. g Cells with eS26 lacking ESS1 or ESS2 accumulate immature 20S pre-rRNA in the cytoplasm. Localization of 20S pre-rRNA in P GAL1 - RPS26Arps26b∆ cells transformed with indicated plasmids was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. h Overexpression of ProtA-FLAG-eS26 is toxic in yeast. The WT yeast strain (BY4741) was transformed with ProtA-FLAG-eS26 or ProtA-FLAG-eS26 lacking ESS2, spotted in 10-fold dilutions on galactose containing media and grown at 25 °C for 4 days. i FLAG-ESS2 fusion protein co-precipitates Tsr2. ESS2 was purified using ProteinA-Tev-FLAG tag, the FLAG eluate was TCA precipitated, separated by SDS-PAGE, and analyzed by Coomassie staining and western analyses using the indicated antibodies
Figure Legend Snippet: Eukaryotic-specific segments of eS26 are required to bind Tsr2. a XL-MS reveals crosslinks between ESS2 and N-terminal domain of Tsr2. The crosslinked residues are listed in the Supplementary Table 1 . b Phylogenetic analyses for eS26 and Tsr2. ESS1, ESS2 from eS26 and Tsr2 are present only in eukaryotes. c Sequence alignment of yeast S26 compared to the indicated species. 70 d ESSs in eS26 are required to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant WT eS26, eS26 deficient in ESS1 and/or ESS2 or archaeal eS26 from Sulfolobus solfataricus . Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining. L = input (1:10 diluted). e Residues 99–109 in eS26-ESS2 are necessary to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with an E. coli lysate containing recombinant WT eS26 or eS26 with variant truncations in C-terminal ESS2. Samples were analyzed as in d . Results from in vitro binding were quantified using ImageJ. f ESS1 and ESS2 deletion from eS26 causes slow growth phenotype in yeast. The conditional P GAL1 - RPS26Arps26b∆ strain was transformed with WT or the indicated truncations of eS26 and spotted in 10-fold dilutions on repressive glucose containing media and grown at 25 °C for 4 days. g Cells with eS26 lacking ESS1 or ESS2 accumulate immature 20S pre-rRNA in the cytoplasm. Localization of 20S pre-rRNA in P GAL1 - RPS26Arps26b∆ cells transformed with indicated plasmids was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. h Overexpression of ProtA-FLAG-eS26 is toxic in yeast. The WT yeast strain (BY4741) was transformed with ProtA-FLAG-eS26 or ProtA-FLAG-eS26 lacking ESS2, spotted in 10-fold dilutions on galactose containing media and grown at 25 °C for 4 days. i FLAG-ESS2 fusion protein co-precipitates Tsr2. ESS2 was purified using ProteinA-Tev-FLAG tag, the FLAG eluate was TCA precipitated, separated by SDS-PAGE, and analyzed by Coomassie staining and western analyses using the indicated antibodies

Techniques Used: Mass Spectrometry, Sequencing, In Vitro, Incubation, Recombinant, SDS Page, Staining, Variant Assay, Binding Assay, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Over Expression, Purification, FLAG-tag, Western Blot

ESSs mediate Tsr2-dependent importin:eS26 complex disassembly. a Tsr2 cannot efficiently dissociate the Kap123:eS26ΔESS1ΔESS2 FLAG complex. The complex of GST-Kap123 with eS26 FLAG , eS26ΔESS1 FLAG eS26ΔESS2 FLAG or eS26ΔESS1ΔESS2 FLAG was immobilized on Glutathione Sepharose and incubated with either buffer alone or with Tsr2 before pull-down. L = input (1:10 diluted). b Tsr2 DWI mutant does not dissociate a Kap123:eS26 complex. The complex of GST-Kap123 with eS26 FLAG was incubated with either buffer alone, purified Tsr2 or Tsr2 DWI before pull-down. L = input (1:10 diluted). c Efficient recruitment of eS26 to pre-40S requires both of the ESSs. Enp1-TAP was isolated from P GAL1 - RPS26Arps26b∆ strain transformed with WT eS26 or eS26 lacking ESS1 or ESS2. After tandem affinity purification, eluates were separated by 4–12% gradient SDS-PAGE and subjected to western analyses using indicated antibodies. Protein levels of uS7, uS3 served as a loading control. d Protein levels of eS26, eS26ΔESS1, eS26ΔESS2 and eS26ΔESS1ΔESS2 in whole-cell extracts of P GAL1 - RPS26Arps26b∆ strain were determined by western analyses using α-eS26 antibodies. Gsp1 protein levels served as a loading control
Figure Legend Snippet: ESSs mediate Tsr2-dependent importin:eS26 complex disassembly. a Tsr2 cannot efficiently dissociate the Kap123:eS26ΔESS1ΔESS2 FLAG complex. The complex of GST-Kap123 with eS26 FLAG , eS26ΔESS1 FLAG eS26ΔESS2 FLAG or eS26ΔESS1ΔESS2 FLAG was immobilized on Glutathione Sepharose and incubated with either buffer alone or with Tsr2 before pull-down. L = input (1:10 diluted). b Tsr2 DWI mutant does not dissociate a Kap123:eS26 complex. The complex of GST-Kap123 with eS26 FLAG was incubated with either buffer alone, purified Tsr2 or Tsr2 DWI before pull-down. L = input (1:10 diluted). c Efficient recruitment of eS26 to pre-40S requires both of the ESSs. Enp1-TAP was isolated from P GAL1 - RPS26Arps26b∆ strain transformed with WT eS26 or eS26 lacking ESS1 or ESS2. After tandem affinity purification, eluates were separated by 4–12% gradient SDS-PAGE and subjected to western analyses using indicated antibodies. Protein levels of uS7, uS3 served as a loading control. d Protein levels of eS26, eS26ΔESS1, eS26ΔESS2 and eS26ΔESS1ΔESS2 in whole-cell extracts of P GAL1 - RPS26Arps26b∆ strain were determined by western analyses using α-eS26 antibodies. Gsp1 protein levels served as a loading control

Techniques Used: Incubation, Mutagenesis, Purification, Isolation, Transformation Assay, Affinity Purification, SDS Page, Western Blot

8) Product Images from "The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein"

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm614

Rrp47p is a multimeric complex. ( A ) Recombinant His( 6 )-Rrp47p purification. Samples were resolved through a 15% SDS–PAGE gel and visualized with Coomassie blue G250. Lane 1, non-induced cell extract; lane 2, cell extract after 4 h induction; lane 3, Ni-NTA superflow eluate; lane 4, SP-sepharose non-bound fraction, lane 5, SP-sepharose eluate; lane 6, peak fraction from the superdex 200 GF column. The positions of molecular weight markers (in kDa) are indicated on the left. ( B ) Gel filtration analysis of the SP-sepharose retained fraction. The A 280 profile is shown, together with the elution volumes of the markers thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.3 kDa). The calibration curve obtained from the molecular weight markers is shown.
Figure Legend Snippet: Rrp47p is a multimeric complex. ( A ) Recombinant His( 6 )-Rrp47p purification. Samples were resolved through a 15% SDS–PAGE gel and visualized with Coomassie blue G250. Lane 1, non-induced cell extract; lane 2, cell extract after 4 h induction; lane 3, Ni-NTA superflow eluate; lane 4, SP-sepharose non-bound fraction, lane 5, SP-sepharose eluate; lane 6, peak fraction from the superdex 200 GF column. The positions of molecular weight markers (in kDa) are indicated on the left. ( B ) Gel filtration analysis of the SP-sepharose retained fraction. The A 280 profile is shown, together with the elution volumes of the markers thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.3 kDa). The calibration curve obtained from the molecular weight markers is shown.

Techniques Used: Recombinant, Purification, SDS Page, Molecular Weight, Filtration

Residues C-terminal to the PMC2NT domain contribute to Rrp47p interaction. ( A ) Schematic of the N-terminal region of Rrp6p. The location of the PMC2NT domain and the four short α-helices α1–α4 are shown. Residues P176 and L197 define the C-termini of truncated Rrp6p constructs. These residues are mutated to stop codons and therefore not the C-terminal residues. ( B ) Pull-down assay of His( 6 )-Rrp47p with GST-tagged N-terminal Rrp6p fragments. Eluates from glutathione-sepharose beads were resolved by SDS–PAGE and assayed by western blot analyses with primary antisera against GST (upper panel) or the His tag (lower panel). Electrophoretic mobilites of protein molecular weight markers (in kDa) are given on the left.
Figure Legend Snippet: Residues C-terminal to the PMC2NT domain contribute to Rrp47p interaction. ( A ) Schematic of the N-terminal region of Rrp6p. The location of the PMC2NT domain and the four short α-helices α1–α4 are shown. Residues P176 and L197 define the C-termini of truncated Rrp6p constructs. These residues are mutated to stop codons and therefore not the C-terminal residues. ( B ) Pull-down assay of His( 6 )-Rrp47p with GST-tagged N-terminal Rrp6p fragments. Eluates from glutathione-sepharose beads were resolved by SDS–PAGE and assayed by western blot analyses with primary antisera against GST (upper panel) or the His tag (lower panel). Electrophoretic mobilites of protein molecular weight markers (in kDa) are given on the left.

Techniques Used: Construct, Pull Down Assay, SDS Page, Western Blot, Molecular Weight

Rrp47p interacts with nucleic acid when bound to Rrp6p. Glutathione-sepharose beads charged with GST or a GST fusion protein containing the N-terminal region of Rrp6p (GST-Rrp6Δ212-721) were incubated with lysate containing His( 6 )-Rrp47p or a control extract (pRSETB). After washing the beads, protein complexes were incubated with radiolabelled E. coli tRNA Phe or a DNA restriction fragment. The non-bound material was removed and bound tRNA or DNA was determined by quantifying the associated Cherenkov radiation. ( A ) Levels of tRNA retained. ( B ) Levels of DNA retained. The results shown are the average of three independent assays, the error bars indicating the associated range.
Figure Legend Snippet: Rrp47p interacts with nucleic acid when bound to Rrp6p. Glutathione-sepharose beads charged with GST or a GST fusion protein containing the N-terminal region of Rrp6p (GST-Rrp6Δ212-721) were incubated with lysate containing His( 6 )-Rrp47p or a control extract (pRSETB). After washing the beads, protein complexes were incubated with radiolabelled E. coli tRNA Phe or a DNA restriction fragment. The non-bound material was removed and bound tRNA or DNA was determined by quantifying the associated Cherenkov radiation. ( A ) Levels of tRNA retained. ( B ) Levels of DNA retained. The results shown are the average of three independent assays, the error bars indicating the associated range.

Techniques Used: Incubation

Rrp47p binds directly to the N-terminal region of Rrp6p. ( A ) Rrp47p co-purifies with Rrp6p in pull-down assays. Glutathione-sepharose beads were incubated first with lysate containing recombinant GST–Rrp6p or GST and then with lysate from cells expressing His( 6 )-Rrp47p or containing the control vector pRSETB. Eluates were resolved through a 10% SDS–PAGE gel and analysed by western blotting using an antibody specific for the His tag. O denotes the top of the resolving gel; X marks the position of the bromophenol blue tracking dye. ( B ) Schematic of the domain structure of Rrp6p. PMC2NT, exonuclease (EXO) and HRDC domains are indicated as boxes. Deletions within Rrp6p constructs made in this study are indicated by broken lines. ( C and D ) Western analyses of eluates from pull-down assays performed using different Rrp6p constructs, as in A. Upper panels show western blots of eluates using an anti-GST antibody; lower panels show corresponding results with an anti-His antibody. Multiple bands are visible with the anti-GST antibody due to proteolytic degradation of Rrp6p. The electrophoretic migration of the full-length Rrp6p protein constructs are indicated to the right of the panels.
Figure Legend Snippet: Rrp47p binds directly to the N-terminal region of Rrp6p. ( A ) Rrp47p co-purifies with Rrp6p in pull-down assays. Glutathione-sepharose beads were incubated first with lysate containing recombinant GST–Rrp6p or GST and then with lysate from cells expressing His( 6 )-Rrp47p or containing the control vector pRSETB. Eluates were resolved through a 10% SDS–PAGE gel and analysed by western blotting using an antibody specific for the His tag. O denotes the top of the resolving gel; X marks the position of the bromophenol blue tracking dye. ( B ) Schematic of the domain structure of Rrp6p. PMC2NT, exonuclease (EXO) and HRDC domains are indicated as boxes. Deletions within Rrp6p constructs made in this study are indicated by broken lines. ( C and D ) Western analyses of eluates from pull-down assays performed using different Rrp6p constructs, as in A. Upper panels show western blots of eluates using an anti-GST antibody; lower panels show corresponding results with an anti-His antibody. Multiple bands are visible with the anti-GST antibody due to proteolytic degradation of Rrp6p. The electrophoretic migration of the full-length Rrp6p protein constructs are indicated to the right of the panels.

Techniques Used: Incubation, Recombinant, Expressing, Plasmid Preparation, SDS Page, Western Blot, Construct, Migration

Rrp47p interacts with the N-terminal region of Rrp6p in yeast. ( A–D ) Western analyses of cell extracts from strains expressing epitope-tagged forms of Rrp47p and/or Rrp6p. Extracts were resolved through 10% SDS–PAGE gels, protein transferred to western blots and the fusion proteins detected using PAP or GST-specific antisera. Relevant strain genotypes are indicated above each lane. The rrp47-zz, rrp6-TAP and zz-rrp6 alleles encode full-length fusion proteins, whereas the zz-Rrp6Δ1-213 and the GST-Rrp6Δ212-721 proteins are truncated Rrp6p variants. The GST fusion proteins were expressed under the control of the GAL promoter; panel D shows analyses of extracts from strains grown in glucose-based media (glc) and in galactose-based media (gal). The upper panel in D shows a western analysis using the GST-specific antibody. The centre panel in D shows a western analysis of the same samples using the PAP antibody. Bands corresponding to the detected fusion proteins are indicated on the right. SDS–PAGE analyses (lower panels) indicate the relative loading of each extract. ( E ) Western analysis of lysates from isogenic wild-type and rrp47- Δ strains, using a His( 6 )-Rrp47p antiserum. Proteins were resolved through a 15% SDS–PAGE gel. The migration of size markers (in kDa) is indicated on the left. A strong band of the expected size is detected in the wild-type lysate (lane 1) and absent in the rrp47-Δ extract (lane 2). ( F ) Western analyses of the bound fractions of extracts from strains expressing GST (lane 1) or GST-Rrp6Δ212-721 (lane 2) after incubation with glutathione-sepharose beads. The upper panel shows a blot probed with the GST-specific antibody, the lower panel shows a blot probed with the His( 6 )-Rrp47p antiserum.
Figure Legend Snippet: Rrp47p interacts with the N-terminal region of Rrp6p in yeast. ( A–D ) Western analyses of cell extracts from strains expressing epitope-tagged forms of Rrp47p and/or Rrp6p. Extracts were resolved through 10% SDS–PAGE gels, protein transferred to western blots and the fusion proteins detected using PAP or GST-specific antisera. Relevant strain genotypes are indicated above each lane. The rrp47-zz, rrp6-TAP and zz-rrp6 alleles encode full-length fusion proteins, whereas the zz-Rrp6Δ1-213 and the GST-Rrp6Δ212-721 proteins are truncated Rrp6p variants. The GST fusion proteins were expressed under the control of the GAL promoter; panel D shows analyses of extracts from strains grown in glucose-based media (glc) and in galactose-based media (gal). The upper panel in D shows a western analysis using the GST-specific antibody. The centre panel in D shows a western analysis of the same samples using the PAP antibody. Bands corresponding to the detected fusion proteins are indicated on the right. SDS–PAGE analyses (lower panels) indicate the relative loading of each extract. ( E ) Western analysis of lysates from isogenic wild-type and rrp47- Δ strains, using a His( 6 )-Rrp47p antiserum. Proteins were resolved through a 15% SDS–PAGE gel. The migration of size markers (in kDa) is indicated on the left. A strong band of the expected size is detected in the wild-type lysate (lane 1) and absent in the rrp47-Δ extract (lane 2). ( F ) Western analyses of the bound fractions of extracts from strains expressing GST (lane 1) or GST-Rrp6Δ212-721 (lane 2) after incubation with glutathione-sepharose beads. The upper panel shows a blot probed with the GST-specific antibody, the lower panel shows a blot probed with the His( 6 )-Rrp47p antiserum.

Techniques Used: Western Blot, Expressing, SDS Page, Gas Chromatography, Migration, Incubation

9) Product Images from "Acetylation of TAFI68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription"

Article Title: Acetylation of TAFI68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription

Journal: The EMBO Journal

doi: 10.1093/emboj/20.6.1353

Fig. 1. TTF-I interacts with PCAF. ( A ) Pull-down of cellular HAT activity by TTF-I. A 10 µg aliquot of FLAG-TTF-I bound to 10 µl of M2-agarose beads (lane 3) or M2 beads saturated with the FLAG peptide (lane 4) was incubated with 2 mg of mouse whole-cell extract proteins for 4 h at 4°C in a total volume of 380 µl. After stringent washing, 50% of the beads were assayed for HAT activity using 5 µg of histones and 1 µCi of [ 3 H]acetyl-CoA. In lanes 1 and 2, histone acetylation by recombinant PCAF or cell extract is shown. Histones were separated by 15% SDS–PAGE and visualized by Coomassie Blue staining (left panel) and fluorography (lanes 1–4). ( B ) Interaction of PCAF with bead-bound TTF-I. A 35 µl aliquot of Ni 2+ -NTA–agarose saturated with histidine-tagged TTF-I (lane 1) or cyclin A (lane 2), and M2-agarose saturated with FLAG-UBF (lane 3) were incubated with extracts from mouse cells. After washing, bead-bound proteins were subjected to western blot analysis using anti-PCAF antibodies. ( C ) Association of cellular TTF-I with PCAF. Bead-bound FLAG-PCAF (lane 2) or control beads (lane 3) were incubated with extract from mouse cells, and associated TTF-I was identified on immunoblots. In lane 1, the amount of TTF-I present in 10% of the extract is shown. ( D ) Co-immunoprecipitation of TTF-I and PCAF. Histidine-tagged TTF-I or cyclin A was co-expressed with FLAG-PCAF in Sf9 cells. PCAF was precipitated with anti-FLAG antibodies (M2) and analyzed on western blots for the presence of TTF-I (lanes 1 and 2) or cyclin A (lanes 3 and 4).
Figure Legend Snippet: Fig. 1. TTF-I interacts with PCAF. ( A ) Pull-down of cellular HAT activity by TTF-I. A 10 µg aliquot of FLAG-TTF-I bound to 10 µl of M2-agarose beads (lane 3) or M2 beads saturated with the FLAG peptide (lane 4) was incubated with 2 mg of mouse whole-cell extract proteins for 4 h at 4°C in a total volume of 380 µl. After stringent washing, 50% of the beads were assayed for HAT activity using 5 µg of histones and 1 µCi of [ 3 H]acetyl-CoA. In lanes 1 and 2, histone acetylation by recombinant PCAF or cell extract is shown. Histones were separated by 15% SDS–PAGE and visualized by Coomassie Blue staining (left panel) and fluorography (lanes 1–4). ( B ) Interaction of PCAF with bead-bound TTF-I. A 35 µl aliquot of Ni 2+ -NTA–agarose saturated with histidine-tagged TTF-I (lane 1) or cyclin A (lane 2), and M2-agarose saturated with FLAG-UBF (lane 3) were incubated with extracts from mouse cells. After washing, bead-bound proteins were subjected to western blot analysis using anti-PCAF antibodies. ( C ) Association of cellular TTF-I with PCAF. Bead-bound FLAG-PCAF (lane 2) or control beads (lane 3) were incubated with extract from mouse cells, and associated TTF-I was identified on immunoblots. In lane 1, the amount of TTF-I present in 10% of the extract is shown. ( D ) Co-immunoprecipitation of TTF-I and PCAF. Histidine-tagged TTF-I or cyclin A was co-expressed with FLAG-PCAF in Sf9 cells. PCAF was precipitated with anti-FLAG antibodies (M2) and analyzed on western blots for the presence of TTF-I (lanes 1 and 2) or cyclin A (lanes 3 and 4).

Techniques Used: HAT Assay, Activity Assay, Incubation, Recombinant, SDS Page, Staining, Western Blot, Immunoprecipitation

Fig. 2. The C-terminal part of TTF-I interacts with PCAF. N-terminal His-tagged deletion mutants TTFΔN185 (lanes 1, 4 and 7), TTFΔN323 (lanes 2, 5 and 8) and TTFΔN445 (lanes 3, 6 and 9) were expressed in Sf9 cells in the presence (lanes 1–3 and 7–9) or absence (lanes 4–6) of FLAG-PCAF. PCAF was bound to M2-agarose by immunoadsorption, and associated TTF-I was identified on immunoblots with anti-TTF-I antibodies. In lanes 1–3, the amount of TTF-I present in 5% of the cell lysates is shown. A schematic representation of TTF-I is shown above. The negative regulatory domain (NRD) and the DNA-binding domain are indicated. The numbers mark amino acid positions.
Figure Legend Snippet: Fig. 2. The C-terminal part of TTF-I interacts with PCAF. N-terminal His-tagged deletion mutants TTFΔN185 (lanes 1, 4 and 7), TTFΔN323 (lanes 2, 5 and 8) and TTFΔN445 (lanes 3, 6 and 9) were expressed in Sf9 cells in the presence (lanes 1–3 and 7–9) or absence (lanes 4–6) of FLAG-PCAF. PCAF was bound to M2-agarose by immunoadsorption, and associated TTF-I was identified on immunoblots with anti-TTF-I antibodies. In lanes 1–3, the amount of TTF-I present in 5% of the cell lysates is shown. A schematic representation of TTF-I is shown above. The negative regulatory domain (NRD) and the DNA-binding domain are indicated. The numbers mark amino acid positions.

Techniques Used: Western Blot, Binding Assay

Fig. 8. mSir2a deacetylates TAF I 68 and decreases rDNA transcription. ( A ) mSir2a deacetylates TAF I 68. FLAG-TAF I 68 and FLAG-PCAF were co-expressed in Sf9 cells and immunopurified with α-FLAG antibodies. A 500 ng aliquot of purified protein FLAG-TAF I 68 was incubated with 200 ng of recombinant mSir2a in the absence (lanes 1–4) or presence (lanes 5–8) of 1 mM NAD + at 30°C for the times indicated. Reactions were subjected to 8% SDS–PAGE, and acetylated proteins were detected on immunoblots using α-acetyl-lysine antibodies. ( B ) Deacetylation of TAF I 68 in the TBP–TAF complex. Recombinant TIF-IB was purified from Sf9 cells that co-expressed the three TAF I s, TBP and PCAF. A 40 ng aliquot of immunopurified complexes was incubated with 200 ng of mSir2 in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of 1 mM NAD + , subjected to 11% SDS–PAGE, and acetylated proteins were visualized on immunoblots using α-acetyl-lysine antibodies. ( C ) Deacetylation of TAF I 68 is reversed by PCAF. Acetylated TAF I 68 was incubated with mSir2a immobilized on Ni + -NTA–agarose for 60 min at 30°C in the absence (lane 1) or presence (lane 2) of 1 mM NAD + . Bead-bound Sir2a was removed by centrifugation, and an aliquot of the NAD + -treated reaction was incubated with 500 ng of GST–PCAF and 10 µM acetyl-CoA (lane 3). Acetylated proteins were detected on immunoblots with α-acetyl-lysine antibodies. ( D ) Deacetylation by Sir2p decreases rDNA transcription in vitro . Transcription factors and RNA polymerase I were pre-incubated for 60 min at 30°C with 200 ng of purified mSir2 immobilized on Ni + -NTA–agarose in the presence (lane 2) or absence (lane 3) of 1 mM NAD + . Bead-bound mSir2a was removed, and the transcriptional activity of the supernatant was measured in the presence of ribonucleotides and template DNA. In lane 1, NAD + was added to the transcription reaction after the pre-incubation step.
Figure Legend Snippet: Fig. 8. mSir2a deacetylates TAF I 68 and decreases rDNA transcription. ( A ) mSir2a deacetylates TAF I 68. FLAG-TAF I 68 and FLAG-PCAF were co-expressed in Sf9 cells and immunopurified with α-FLAG antibodies. A 500 ng aliquot of purified protein FLAG-TAF I 68 was incubated with 200 ng of recombinant mSir2a in the absence (lanes 1–4) or presence (lanes 5–8) of 1 mM NAD + at 30°C for the times indicated. Reactions were subjected to 8% SDS–PAGE, and acetylated proteins were detected on immunoblots using α-acetyl-lysine antibodies. ( B ) Deacetylation of TAF I 68 in the TBP–TAF complex. Recombinant TIF-IB was purified from Sf9 cells that co-expressed the three TAF I s, TBP and PCAF. A 40 ng aliquot of immunopurified complexes was incubated with 200 ng of mSir2 in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of 1 mM NAD + , subjected to 11% SDS–PAGE, and acetylated proteins were visualized on immunoblots using α-acetyl-lysine antibodies. ( C ) Deacetylation of TAF I 68 is reversed by PCAF. Acetylated TAF I 68 was incubated with mSir2a immobilized on Ni + -NTA–agarose for 60 min at 30°C in the absence (lane 1) or presence (lane 2) of 1 mM NAD + . Bead-bound Sir2a was removed by centrifugation, and an aliquot of the NAD + -treated reaction was incubated with 500 ng of GST–PCAF and 10 µM acetyl-CoA (lane 3). Acetylated proteins were detected on immunoblots with α-acetyl-lysine antibodies. ( D ) Deacetylation by Sir2p decreases rDNA transcription in vitro . Transcription factors and RNA polymerase I were pre-incubated for 60 min at 30°C with 200 ng of purified mSir2 immobilized on Ni + -NTA–agarose in the presence (lane 2) or absence (lane 3) of 1 mM NAD + . Bead-bound mSir2a was removed, and the transcriptional activity of the supernatant was measured in the presence of ribonucleotides and template DNA. In lane 1, NAD + was added to the transcription reaction after the pre-incubation step.

Techniques Used: Purification, Incubation, Recombinant, SDS Page, Western Blot, Centrifugation, In Vitro, Activity Assay

Fig. 3. PCAF acetylates TAF I 68 in vitro . ( A ) Acetylation of recombinant proteins. A 2 µg aliquot of the proteins indicated was incubated with 500 ng of FLAG-PCAF, 1 µCi of [ 3 H]acetyl-CoA and 0.4 µM TSA in a total volume of 30 µl of buffer AM-100 for 30 min at 30°C. Proteins were separated by 10% SDS–PAGE, and acetylated proteins were visualized by fluorography. ( B ) TAF I 68 interacts with PCAF. GST–PCAF or GST were bound to glutathione–agarose beads and incubated with 35 S-labeled TAF I 68 (lanes 2 and 3), TTFΔN323 (lanes 5 and 6) or UBF (lanes 8 and 9). Bound proteins were analyzed by 8% SDS–PAGE and autoradiography. Ten percent of the 35 S-labeled input proteins are shown in lanes 1, 4 and 7. ( C ) Acetylation of TAF I 68 with CBP, GCN5 and PCAF. A 500 ng aliquot of TAF I 68 was incubated for 30 min at 30°C with 1 µCi of [ 3 H]acetyl-CoA, 0.4 µM TSA and comparable units of HAT activity of CBP (lane 2), GCN5 (lane 3) or PCAF (lane 4). After gel electrophoresis, acetylated TAF I 68 was visualized by fluorography. A Coomassie Blue stain of 500 ng of TAF I 68 is shown on the left.
Figure Legend Snippet: Fig. 3. PCAF acetylates TAF I 68 in vitro . ( A ) Acetylation of recombinant proteins. A 2 µg aliquot of the proteins indicated was incubated with 500 ng of FLAG-PCAF, 1 µCi of [ 3 H]acetyl-CoA and 0.4 µM TSA in a total volume of 30 µl of buffer AM-100 for 30 min at 30°C. Proteins were separated by 10% SDS–PAGE, and acetylated proteins were visualized by fluorography. ( B ) TAF I 68 interacts with PCAF. GST–PCAF or GST were bound to glutathione–agarose beads and incubated with 35 S-labeled TAF I 68 (lanes 2 and 3), TTFΔN323 (lanes 5 and 6) or UBF (lanes 8 and 9). Bound proteins were analyzed by 8% SDS–PAGE and autoradiography. Ten percent of the 35 S-labeled input proteins are shown in lanes 1, 4 and 7. ( C ) Acetylation of TAF I 68 with CBP, GCN5 and PCAF. A 500 ng aliquot of TAF I 68 was incubated for 30 min at 30°C with 1 µCi of [ 3 H]acetyl-CoA, 0.4 µM TSA and comparable units of HAT activity of CBP (lane 2), GCN5 (lane 3) or PCAF (lane 4). After gel electrophoresis, acetylated TAF I 68 was visualized by fluorography. A Coomassie Blue stain of 500 ng of TAF I 68 is shown on the left.

Techniques Used: In Vitro, Recombinant, Incubation, SDS Page, Labeling, Autoradiography, HAT Assay, Activity Assay, Nucleic Acid Electrophoresis, Staining

10) Product Images from "Drosophila doubletime Mutations Which either Shorten or Lengthen the Period of Circadian Rhythms Decrease the Protein Kinase Activity of Casein Kinase I"

Article Title: Drosophila doubletime Mutations Which either Shorten or Lengthen the Period of Circadian Rhythms Decrease the Protein Kinase Activity of Casein Kinase I

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.2.886-898.2004

Bacterially expressed CKIδ protein from Xenopus phosphorylates casein and is sensitive to a CKI-specific inhibitor. Recombinant CKIδ protein from Xenopus and a modified S-tag, to which CKIδ was fused to produce the full-length CKIδ (see Materials and Methods), were expressed in bacteria and purified on S-protein agarose. The agarose beads with the indicated purified protein were incubated with [γ- 32 P]ATP, with or without (+ or −) casein for 5 min at 37°C, and aliquots of the reactions were analyzed by SDS-PAGE, followed by silver staining of the gel (A), by detection of CKIδ with an antibody (B), or by autoradiography to detect the phosphorylated products (C). Chemiluminescent signals arising from immunoblot detection of CKIδ were clearly distinguishable from kinase-produced 32 P signals for two reasons: the chemiluminescent signals disappeared after several hours while the radioactive signals persisted, and direct autoradiography or phosphorimager analysis of the gels detected only the radioactive signal. Moreover, chemiluminescent exposure times (e.g., B) were typically too short (less than a minute) to detect the 32 P signal. The star denotes a non-CKI antigen which always copurified with CKIδ. Recombinant CKIδ phosphorylates casein and itself, while the modified S-tag does not produce any kinase activity. Ovalbumin (OVA) was present in the reaction buffer. (D) Bacterially expressed CKIδ was assayed under the same conditions as in panels A to C, except that the CKI-specific inhibitor CKI-7 was present at 50 μM and the ATP concentration was 20 μM. An autoradiograph is shown, with the mobilities of the phosphorylation products indicated on the left. CKI-7 produced a dramatic reduction (to 12% of uninhibited levels) in activity in comparison with the normal reaction conditions, as well as in comparison with reactions containing the CKI-7 vehicle (DMSO) alone.
Figure Legend Snippet: Bacterially expressed CKIδ protein from Xenopus phosphorylates casein and is sensitive to a CKI-specific inhibitor. Recombinant CKIδ protein from Xenopus and a modified S-tag, to which CKIδ was fused to produce the full-length CKIδ (see Materials and Methods), were expressed in bacteria and purified on S-protein agarose. The agarose beads with the indicated purified protein were incubated with [γ- 32 P]ATP, with or without (+ or −) casein for 5 min at 37°C, and aliquots of the reactions were analyzed by SDS-PAGE, followed by silver staining of the gel (A), by detection of CKIδ with an antibody (B), or by autoradiography to detect the phosphorylated products (C). Chemiluminescent signals arising from immunoblot detection of CKIδ were clearly distinguishable from kinase-produced 32 P signals for two reasons: the chemiluminescent signals disappeared after several hours while the radioactive signals persisted, and direct autoradiography or phosphorimager analysis of the gels detected only the radioactive signal. Moreover, chemiluminescent exposure times (e.g., B) were typically too short (less than a minute) to detect the 32 P signal. The star denotes a non-CKI antigen which always copurified with CKIδ. Recombinant CKIδ phosphorylates casein and itself, while the modified S-tag does not produce any kinase activity. Ovalbumin (OVA) was present in the reaction buffer. (D) Bacterially expressed CKIδ was assayed under the same conditions as in panels A to C, except that the CKI-specific inhibitor CKI-7 was present at 50 μM and the ATP concentration was 20 μM. An autoradiograph is shown, with the mobilities of the phosphorylation products indicated on the left. CKI-7 produced a dramatic reduction (to 12% of uninhibited levels) in activity in comparison with the normal reaction conditions, as well as in comparison with reactions containing the CKI-7 vehicle (DMSO) alone.

Techniques Used: Recombinant, Modification, Purification, Incubation, SDS Page, Silver Staining, Autoradiography, Produced, Activity Assay, Concentration Assay

The dbt S and dbt L mutations reduced the protein kinase activity of bacterially expressed CKIδ from Xenopus . The dbt L and dbt S mutations were introduced into ckI δ by site-directed mutagenesis. (A and B) S-tag wild-type CKIδ (CKIδ WT ), CKIδ L , and CKIδ S were purified on S-protein agarose, incubated with casein and [γ 32 -P]ATP for 5 min at 37°C, and analyzed by SDS-PAGE of reaction aliquots, followed by detection of CKIδ with an antibody (B) or detection of phosphorylated products by autoradiography (A). The concentration of casein (in milligrams per milliliter) is shown at the top (replicates of the 0.38-mg/ml assay are shown.). The mutant CKI proteins produced less phosphorylation of casein, despite the reduced levels of wild-type CKI detected by immunoblot analysis. (B and D) S-tag wild-type CKIδ, CKIδ S , and CKIδ L were purified on S-protein agarose, incubated with [γ 32 -P]ATP with (+) or without (−) GST-PER protein (PERIOD) for 5 min at 37°C, and reaction aliquots were analyzed by SDS-PAGE, followed by either immunoblot analysis to detect CKIδ (D) or autoradiography to detect the reaction products (C). The mutant CKIδs phosphorylate PER less efficiently than the wild type enzyme, since there are higher amounts of mutant enzymes in the reactions.
Figure Legend Snippet: The dbt S and dbt L mutations reduced the protein kinase activity of bacterially expressed CKIδ from Xenopus . The dbt L and dbt S mutations were introduced into ckI δ by site-directed mutagenesis. (A and B) S-tag wild-type CKIδ (CKIδ WT ), CKIδ L , and CKIδ S were purified on S-protein agarose, incubated with casein and [γ 32 -P]ATP for 5 min at 37°C, and analyzed by SDS-PAGE of reaction aliquots, followed by detection of CKIδ with an antibody (B) or detection of phosphorylated products by autoradiography (A). The concentration of casein (in milligrams per milliliter) is shown at the top (replicates of the 0.38-mg/ml assay are shown.). The mutant CKI proteins produced less phosphorylation of casein, despite the reduced levels of wild-type CKI detected by immunoblot analysis. (B and D) S-tag wild-type CKIδ, CKIδ S , and CKIδ L were purified on S-protein agarose, incubated with [γ 32 -P]ATP with (+) or without (−) GST-PER protein (PERIOD) for 5 min at 37°C, and reaction aliquots were analyzed by SDS-PAGE, followed by either immunoblot analysis to detect CKIδ (D) or autoradiography to detect the reaction products (C). The mutant CKIδs phosphorylate PER less efficiently than the wild type enzyme, since there are higher amounts of mutant enzymes in the reactions.

Techniques Used: Activity Assay, Mutagenesis, Purification, Incubation, SDS Page, Autoradiography, Concentration Assay, Produced

GST pull-down assays demonstrate that the N-terminal 292 amino acids of DBT contain the interaction site for PER. GST-DBT fusions of the indicated genotype (GST, GST without DBT; F, GST fused with full-length DBT [GST-DBT]; N, GST fused with amino acids 1 to 292 of DBT [GST-DBTN]; C, GST fused with amino acids 293 to 440 of DBT [GST-DBTC]) were purified on glutathione agarose and incubated with [ 35 S]methionine-labeled PER produced by in vitro-coupled transcription-translation. After any unbound PER was washed off, the material still bound to the agarose was analyzed by SDS-PAGE analysis. Total proteins were visualized by staining the gel with Coomassie brilliant blue (A), while 35 S-labeled proteins were visualized with autoradiography (B). The amount of PER added to the 5× binding assays was 5 times the amount added to the 1× assays. The 1× assays were incubated for 2 h at room temperature, while the 5× assays were incubated for 5 min at room temperature. In order from the top of the gel in panel A, the markers indicate the mobilities of the full-length GST-DBT fusion protein (triangle), the GST-DBTN fusion protein (asterisk), ovalbumin added to the binding buffer to prevent nonspecific binding (square), the GST-DBTC fusion protein (circle), and GST protein (diamond). The open arrow in panel B indicates bound PER. Note that PER is bound specifically by full-length GST-DBT and GST-DBTN, but not by GST or GST-DBTC, despite the much higher levels of GST and GST-DBTC proteins.
Figure Legend Snippet: GST pull-down assays demonstrate that the N-terminal 292 amino acids of DBT contain the interaction site for PER. GST-DBT fusions of the indicated genotype (GST, GST without DBT; F, GST fused with full-length DBT [GST-DBT]; N, GST fused with amino acids 1 to 292 of DBT [GST-DBTN]; C, GST fused with amino acids 293 to 440 of DBT [GST-DBTC]) were purified on glutathione agarose and incubated with [ 35 S]methionine-labeled PER produced by in vitro-coupled transcription-translation. After any unbound PER was washed off, the material still bound to the agarose was analyzed by SDS-PAGE analysis. Total proteins were visualized by staining the gel with Coomassie brilliant blue (A), while 35 S-labeled proteins were visualized with autoradiography (B). The amount of PER added to the 5× binding assays was 5 times the amount added to the 1× assays. The 1× assays were incubated for 2 h at room temperature, while the 5× assays were incubated for 5 min at room temperature. In order from the top of the gel in panel A, the markers indicate the mobilities of the full-length GST-DBT fusion protein (triangle), the GST-DBTN fusion protein (asterisk), ovalbumin added to the binding buffer to prevent nonspecific binding (square), the GST-DBTC fusion protein (circle), and GST protein (diamond). The open arrow in panel B indicates bound PER. Note that PER is bound specifically by full-length GST-DBT and GST-DBTN, but not by GST or GST-DBTC, despite the much higher levels of GST and GST-DBTC proteins.

Techniques Used: Purification, Incubation, Labeling, Produced, In Vitro, SDS Page, Staining, Autoradiography, Binding Assay

GST pull-down assays demonstrate that DBT + , DBT S , and DBT L proteins interact equivalently with PER. GST-DBT fusions of the indicated genotype (GST, GST without DBT; W, GST fused with wild-type DBT; S, GST fused with DBT S ; L, GST fused with DBT L ) were purified on glutathione agarose and incubated for 2 h with [ 35 S]methionine-labeled PER or luciferase produced by in vitro-coupled transcription-translation. After any unbound PER or luciferase was washed off, the material still bound to the agarose was analyzed by SDS-PAGE analysis. Total proteins were visualized by staining the gel with Coomassie brilliant blue (A), while 35 S-labeled proteins were visualized with autoradiography (B). The in vitro transcription-translation reactions were electrophoresed directly without binding in the first two lanes. The amount of luciferase in lane 1 was 1.25 times the amount used in the binding assays analyzed here, while the amount of PER in lane 2 was 6.25 times the amount of PER used in the binding assays analyzed here. Star, the GST-DBT fusion protein; Ω, ovalbumin added to the binding buffer to prevent nonspecific binding; diamond, GST protein, incomplete translation of GST-DBT, or breakdown product from GST-DBT; open arrow, bound PER.
Figure Legend Snippet: GST pull-down assays demonstrate that DBT + , DBT S , and DBT L proteins interact equivalently with PER. GST-DBT fusions of the indicated genotype (GST, GST without DBT; W, GST fused with wild-type DBT; S, GST fused with DBT S ; L, GST fused with DBT L ) were purified on glutathione agarose and incubated for 2 h with [ 35 S]methionine-labeled PER or luciferase produced by in vitro-coupled transcription-translation. After any unbound PER or luciferase was washed off, the material still bound to the agarose was analyzed by SDS-PAGE analysis. Total proteins were visualized by staining the gel with Coomassie brilliant blue (A), while 35 S-labeled proteins were visualized with autoradiography (B). The in vitro transcription-translation reactions were electrophoresed directly without binding in the first two lanes. The amount of luciferase in lane 1 was 1.25 times the amount used in the binding assays analyzed here, while the amount of PER in lane 2 was 6.25 times the amount of PER used in the binding assays analyzed here. Star, the GST-DBT fusion protein; Ω, ovalbumin added to the binding buffer to prevent nonspecific binding; diamond, GST protein, incomplete translation of GST-DBT, or breakdown product from GST-DBT; open arrow, bound PER.

Techniques Used: Purification, Incubation, Labeling, Luciferase, Produced, In Vitro, SDS Page, Staining, Autoradiography, Binding Assay

11) Product Images from "AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization"

Article Title: AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization

Journal: Autophagy

doi: 10.4161/auto.20586

Figure 1. ULK1 and ULK2 associate with AMPK. (A) FLAG-AMPKα1 can interact with myc-ULK1. HEK293T cells were cotransfected with the indicated plasmids and FLAG-AMPKα1 was immunoprecipitated from the cell lysates using anti-FLAG agarose beads. Cell lysates and immunoprecipitates were analyzed by western blot with the antibodies indicated. (B) YFP-ULK2 can interact with FLAG-AMPKα1. HEK293T cells were cotransfected and processed as described in (A). (C) Endogeneous AMPKα1 can be detected in myc-ULK1 immunoprecipitates of myc-ULK1 COS7 cell lysates. Lysates from COS7 cells infected with control virus or virus encoding myc-ULK1 were incubated with the indicated antibodies and processed as described in Materials and Methods. Western blots shown were probed with the indicated antibodies. (D) Endogeneous AMPKα1 can be detected in myc-ULK1 immunoprecipitates from myc-ULK1 infected 293T cells. 293T cells were infected with myc-ULK1 encoding virus and lysates from these cells were incubated with the indicated antibodies and immunoprecipitates analyzed by western blot with the indicated antibodies. (E) Endogeneous AMPKα2 can be co-immunoprecipitated with myc-ULK1. Myc-ULK1 293T cells were generated and processed as described in (D). Western blots were probed with the antibodies indicated. (F) Endogeneous AMPKα1 and ULK1 can be co-immunoprecipitated. MEFs were lysed and endogenous AMPKα1 was immunoprecipitated as in (D). Cell lysates and immunoprecipitates were analyzed by western blot with the antibodies indicated. The asterisk in (A, C, D and E) denotes IgG, the asterisk in (B) denotes a nonspecific band.
Figure Legend Snippet: Figure 1. ULK1 and ULK2 associate with AMPK. (A) FLAG-AMPKα1 can interact with myc-ULK1. HEK293T cells were cotransfected with the indicated plasmids and FLAG-AMPKα1 was immunoprecipitated from the cell lysates using anti-FLAG agarose beads. Cell lysates and immunoprecipitates were analyzed by western blot with the antibodies indicated. (B) YFP-ULK2 can interact with FLAG-AMPKα1. HEK293T cells were cotransfected and processed as described in (A). (C) Endogeneous AMPKα1 can be detected in myc-ULK1 immunoprecipitates of myc-ULK1 COS7 cell lysates. Lysates from COS7 cells infected with control virus or virus encoding myc-ULK1 were incubated with the indicated antibodies and processed as described in Materials and Methods. Western blots shown were probed with the indicated antibodies. (D) Endogeneous AMPKα1 can be detected in myc-ULK1 immunoprecipitates from myc-ULK1 infected 293T cells. 293T cells were infected with myc-ULK1 encoding virus and lysates from these cells were incubated with the indicated antibodies and immunoprecipitates analyzed by western blot with the indicated antibodies. (E) Endogeneous AMPKα2 can be co-immunoprecipitated with myc-ULK1. Myc-ULK1 293T cells were generated and processed as described in (D). Western blots were probed with the antibodies indicated. (F) Endogeneous AMPKα1 and ULK1 can be co-immunoprecipitated. MEFs were lysed and endogenous AMPKα1 was immunoprecipitated as in (D). Cell lysates and immunoprecipitates were analyzed by western blot with the antibodies indicated. The asterisk in (A, C, D and E) denotes IgG, the asterisk in (B) denotes a nonspecific band.

Techniques Used: Immunoprecipitation, Western Blot, Infection, Incubation, Generated

Figure 6. AMPK regulates YWHAZ binding to ULK1. COS7 cells infected with myc-ULK1 were treated with (A) 25 mM 2DG; (B) 1 mM AICAR; or (C) EBSS for the times indicated. The 0 min time point indicates treatment with vehicle for the longest timepoint. Cell lysates were incubated with GST-YWHAZ bound to glutathione sepharose beads or plain glutathione sepharose beads as a control. Immunoblots were probed with the antibodies indicated. (D) Wild-type MEFs and MEFs lacking the genes for both ( PRKAA1 −/− ,PRKAA2 −/− ) or one of the PRKAA isoforms ( PRKAA1 −/− and PRKAA2 −/− ) were treated with vehicle (0 min time point) or 25 mM 2DG for 15 min and endogenous ULK1 was pulled down with GST-YWHAZ. The GST-tag alone served as a negative control. Western blot analysis was performed with the antibodies indicated. (E) MEFs stably overexpressing myc-ULK were treated with 25 mM 2DG as in (D) and endogenous YWHAZ was immunoprecipitated using a pan-14–3-3 antibody or nonspecific mouse IgG1 as a negative control. Western blot analysis was performed with the antibodies indicated. (F) Wild-type or nonphosphorylatable serine to alanine mutants of myc-ULK1 at four potential YWHAZ-binding sites, S467A, S494A, S555A and T659A as well as the S555A-T659A double mutant were stably overexpressed in COS7 cells were treated with 25 mM 2DG as in (D). Following lysis, myc-ULK1 variants were pulled down with GST or GST-YWHAZ. Western blot analysis was performed with the antibodies indicated.
Figure Legend Snippet: Figure 6. AMPK regulates YWHAZ binding to ULK1. COS7 cells infected with myc-ULK1 were treated with (A) 25 mM 2DG; (B) 1 mM AICAR; or (C) EBSS for the times indicated. The 0 min time point indicates treatment with vehicle for the longest timepoint. Cell lysates were incubated with GST-YWHAZ bound to glutathione sepharose beads or plain glutathione sepharose beads as a control. Immunoblots were probed with the antibodies indicated. (D) Wild-type MEFs and MEFs lacking the genes for both ( PRKAA1 −/− ,PRKAA2 −/− ) or one of the PRKAA isoforms ( PRKAA1 −/− and PRKAA2 −/− ) were treated with vehicle (0 min time point) or 25 mM 2DG for 15 min and endogenous ULK1 was pulled down with GST-YWHAZ. The GST-tag alone served as a negative control. Western blot analysis was performed with the antibodies indicated. (E) MEFs stably overexpressing myc-ULK were treated with 25 mM 2DG as in (D) and endogenous YWHAZ was immunoprecipitated using a pan-14–3-3 antibody or nonspecific mouse IgG1 as a negative control. Western blot analysis was performed with the antibodies indicated. (F) Wild-type or nonphosphorylatable serine to alanine mutants of myc-ULK1 at four potential YWHAZ-binding sites, S467A, S494A, S555A and T659A as well as the S555A-T659A double mutant were stably overexpressed in COS7 cells were treated with 25 mM 2DG as in (D). Following lysis, myc-ULK1 variants were pulled down with GST or GST-YWHAZ. Western blot analysis was performed with the antibodies indicated.

Techniques Used: Binding Assay, Infection, Incubation, Western Blot, Negative Control, Stable Transfection, Immunoprecipitation, Mutagenesis, Lysis

Figure 2. Association of ULK1 and AMPK is independent of their kinase activities and requires the ULK1 spacer region and the AMPKα subunit. (A) The kinase activity of ULK1 is not required for its association with AMPK. COS7 cells infected with wild-type (wt) or kinase dead (kd) myc-ULK1 were lysed and subjected to immunoprecipitation using an AMPKα2-specific rabbit antibody or non-specific rabbit-anti-mouse IgG (RαM) as a control. Western blot analysis was performed with the antibodies indicated. (B) The kinase domain of AMPK can associate with ULK1 and AMPK kinase activity is not required. HEK293T cells were cotransfected with myc-ULK1 and wild-type, kinase dead or constitutively active (ca) versions of FLAG-AMPKα1 or an empty vector. FLAG-AMPKα1 proteins were immunoprecipitated using anti-FLAG agarose beads. Western blot analysis was performed with the antibodies indicated. (C) The ULK1 spacer region can co-immunoprecipitate AMPK. HEK293T cells were transfected with empty vector or FLAG-tagged ULK1 fragments containing various combinations of the three ULK1 domains depicted in the schematic drawing. ULK1-FLAG proteins were immunoprecipitated using anti-FLAG agarose beads and cell lysates and immunoprecipitates were analyzed by western blot with the antibodies indicated.
Figure Legend Snippet: Figure 2. Association of ULK1 and AMPK is independent of their kinase activities and requires the ULK1 spacer region and the AMPKα subunit. (A) The kinase activity of ULK1 is not required for its association with AMPK. COS7 cells infected with wild-type (wt) or kinase dead (kd) myc-ULK1 were lysed and subjected to immunoprecipitation using an AMPKα2-specific rabbit antibody or non-specific rabbit-anti-mouse IgG (RαM) as a control. Western blot analysis was performed with the antibodies indicated. (B) The kinase domain of AMPK can associate with ULK1 and AMPK kinase activity is not required. HEK293T cells were cotransfected with myc-ULK1 and wild-type, kinase dead or constitutively active (ca) versions of FLAG-AMPKα1 or an empty vector. FLAG-AMPKα1 proteins were immunoprecipitated using anti-FLAG agarose beads. Western blot analysis was performed with the antibodies indicated. (C) The ULK1 spacer region can co-immunoprecipitate AMPK. HEK293T cells were transfected with empty vector or FLAG-tagged ULK1 fragments containing various combinations of the three ULK1 domains depicted in the schematic drawing. ULK1-FLAG proteins were immunoprecipitated using anti-FLAG agarose beads and cell lysates and immunoprecipitates were analyzed by western blot with the antibodies indicated.

Techniques Used: Activity Assay, Infection, Immunoprecipitation, Western Blot, Plasmid Preparation, Transfection

12) Product Images from "UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II"

Article Title: UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200404045

Purification and characterization of Myo2. (A) SDS-PAGE of proteins stained with Coomassie blue. The leftmost lane shows proteins affinity purified from a wild-type strain (MLP 479) overexpressing GST-tagged light chains from the plasmids pGST- cdc4 and pGST- rlc1 . The right five lanes show samples from steps in the purification of Myo2 from a strain (MLP 374) overexpressing Myo2p from the 3nmt1 promoter and GST-tagged light chains from pGST- cdc4 and pGST- rlc1 . The steps are the total cell extract after centrifuging lysed cells at 100,000 g , proteins eluted from glutathione-Sepharose, the products of thrombin cleavage, the pooled peak from gel filtration, and the pooled peak of purified Myo2 from hydroxyapatite chromatography. Polypeptides are named on the right. (B) ATP-sensitive binding of purified Myo2 to actin filaments evaluated by SDS-PAGE and staining with Coomassie blue. Samples containing 0.75 μM Myo2 in 0.5 M NaCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 2 mM MgCl 2 with 0 or 10 μM actin filaments, and 0 or 2 mM ATP were centrifuged at 120,000 g for 45 min. (top) Myo2 remains in the supernatant in absence of actin filaments. (bottom) Myo2 pellets with actin filaments in the absence but not the presence of 2 mM ATP. S, supernatant; P, pellet. (C) Dependence of the solubility of Myo2 on KCl concentration. Samples of 0.5 μM Myo2 in 10 mM imidazole, 1 mM DTT, and 0 to 500 mM KCl were centrifuged at 120,000 g for 10 min. Soluble Myo2 in the supernatant was determined by both the Bradford protein assay and densitometry of samples stained on protein gels. Two independent experiments are shown based on densitometry data.
Figure Legend Snippet: Purification and characterization of Myo2. (A) SDS-PAGE of proteins stained with Coomassie blue. The leftmost lane shows proteins affinity purified from a wild-type strain (MLP 479) overexpressing GST-tagged light chains from the plasmids pGST- cdc4 and pGST- rlc1 . The right five lanes show samples from steps in the purification of Myo2 from a strain (MLP 374) overexpressing Myo2p from the 3nmt1 promoter and GST-tagged light chains from pGST- cdc4 and pGST- rlc1 . The steps are the total cell extract after centrifuging lysed cells at 100,000 g , proteins eluted from glutathione-Sepharose, the products of thrombin cleavage, the pooled peak from gel filtration, and the pooled peak of purified Myo2 from hydroxyapatite chromatography. Polypeptides are named on the right. (B) ATP-sensitive binding of purified Myo2 to actin filaments evaluated by SDS-PAGE and staining with Coomassie blue. Samples containing 0.75 μM Myo2 in 0.5 M NaCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 2 mM MgCl 2 with 0 or 10 μM actin filaments, and 0 or 2 mM ATP were centrifuged at 120,000 g for 45 min. (top) Myo2 remains in the supernatant in absence of actin filaments. (bottom) Myo2 pellets with actin filaments in the absence but not the presence of 2 mM ATP. S, supernatant; P, pellet. (C) Dependence of the solubility of Myo2 on KCl concentration. Samples of 0.5 μM Myo2 in 10 mM imidazole, 1 mM DTT, and 0 to 500 mM KCl were centrifuged at 120,000 g for 10 min. Soluble Myo2 in the supernatant was determined by both the Bradford protein assay and densitometry of samples stained on protein gels. Two independent experiments are shown based on densitometry data.

Techniques Used: Purification, SDS Page, Staining, Affinity Purification, Filtration, Chromatography, Binding Assay, Solubility, Concentration Assay, Bradford Protein Assay

Rng3p stimulates Myo2 activity. (A) Lanes 1–5 show samples from steps in the purification of Myo2 from a strain (MLP 676) containing a chromosomal rng3-GFP 3 fusion overexpressing Myo2p from the 41nmt1 promoter and GST-tagged light chains from pGST- cdc4 and pGST- rlc1 . Samples were run on an SDS-PAGE gel, immunoblotted and probed with Myo2 heavy chain antibodies (top) and GFP antibodies (bottom). (B) Bead binding assay for interaction of chromosomal Rng3p-GST with Myo2. Strains: FY 435 carrying pGST- rlc1 , MLP 694 ( rng3-GST ), MLP 693 ( rng3-GST , myo2-E1 ), and MLP 695 (wild-type). GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain (top) and Rng3p-GST (α-GST; bottom). pGST- rlc1 , positive control with GST-Rlc1p. wild type, negative control lacking a GST fusion. (C) Bead binding assay for interaction of overexpressed GST-Rng3p (pGST- rng3 - FL ) and GST-Rng3-UCS domain (pGST- rng3 - UCS ) with Myo2. Strains: FY 435 (wild-type) and TP 73 ( myo2-E1 ) carrying plasmids pGST- rng3 , pGST- rng3-UCS , pGST- cam1 , and pGST- rlc1 . GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain. pGST- cam1 , negative control with GST-Cam1p. pGST- rlc1 , positive control with GST-Rlc1p. (D) GST-Rng3p purified from S. pombe and recombinant GST-Rng3p purified from Escherichia coli . SDS-PAGE gel stained with Coomassie blue. Lower band in the S. pombe lane represents a breakdown product. (E–J) Actin filament gliding assays. Time-lapse fluorescence micrographs of filaments labeled with rhodamine-phalloidin (also, see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1 ). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Bar, 5 μm. Conditions: indicated concentrations of Myo2 and GST-Rng3p were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Crude one-step purified Myo2 (0.25 mg/ml impure protein). (F) Three-step–purified 75 nM Myo2 preincubated with 250 nM native S. pombe GST-Rng3p. (G) Three-step–purified 75 nM Myo2 preincubated with 250 nM recombinant GST-Rng3p. (H) Three-step–purified 75 nM Myo2 alone. (I) S. pombe GST-Rng3p (250 nM) alone. (J) recombinant GST-Rng3p (250 nM) alone. (K) Dependence of the actin-activated ATPase activity of three-step–purified Myo2 as a function of the concentrations of native (open circles) and recombinant (closed circles) GST-Rng3p. Conditions: 30 nM Myo2 and 10 μM actin filaments in 2 mM ATP, 3 mM MgCl 2 , 0.1 mM CaCl 2 , and 75 mM KCl. Error bars show SD. (L) Dependence of the number of actin filaments captured by two-step–purified Myo2 on the concentration of recombinant GST-Rng3p. All filaments in a 130 μm 2 frame of a fluorescence micrograph were counted. (squares) 20 nM two-step–purified Myo2. No gliding was observed in the absence of GST-Rng3p. (circles) 200 nM two-step–purified Myo2. These samples supported gliding in the absence of GST-Rng3p. (horizontal line) 150 nM crude Myo2 purified after overexpression from MLP 374 ( 3nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). These samples supported robust gliding.
Figure Legend Snippet: Rng3p stimulates Myo2 activity. (A) Lanes 1–5 show samples from steps in the purification of Myo2 from a strain (MLP 676) containing a chromosomal rng3-GFP 3 fusion overexpressing Myo2p from the 41nmt1 promoter and GST-tagged light chains from pGST- cdc4 and pGST- rlc1 . Samples were run on an SDS-PAGE gel, immunoblotted and probed with Myo2 heavy chain antibodies (top) and GFP antibodies (bottom). (B) Bead binding assay for interaction of chromosomal Rng3p-GST with Myo2. Strains: FY 435 carrying pGST- rlc1 , MLP 694 ( rng3-GST ), MLP 693 ( rng3-GST , myo2-E1 ), and MLP 695 (wild-type). GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain (top) and Rng3p-GST (α-GST; bottom). pGST- rlc1 , positive control with GST-Rlc1p. wild type, negative control lacking a GST fusion. (C) Bead binding assay for interaction of overexpressed GST-Rng3p (pGST- rng3 - FL ) and GST-Rng3-UCS domain (pGST- rng3 - UCS ) with Myo2. Strains: FY 435 (wild-type) and TP 73 ( myo2-E1 ) carrying plasmids pGST- rng3 , pGST- rng3-UCS , pGST- cam1 , and pGST- rlc1 . GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain. pGST- cam1 , negative control with GST-Cam1p. pGST- rlc1 , positive control with GST-Rlc1p. (D) GST-Rng3p purified from S. pombe and recombinant GST-Rng3p purified from Escherichia coli . SDS-PAGE gel stained with Coomassie blue. Lower band in the S. pombe lane represents a breakdown product. (E–J) Actin filament gliding assays. Time-lapse fluorescence micrographs of filaments labeled with rhodamine-phalloidin (also, see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1 ). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Bar, 5 μm. Conditions: indicated concentrations of Myo2 and GST-Rng3p were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Crude one-step purified Myo2 (0.25 mg/ml impure protein). (F) Three-step–purified 75 nM Myo2 preincubated with 250 nM native S. pombe GST-Rng3p. (G) Three-step–purified 75 nM Myo2 preincubated with 250 nM recombinant GST-Rng3p. (H) Three-step–purified 75 nM Myo2 alone. (I) S. pombe GST-Rng3p (250 nM) alone. (J) recombinant GST-Rng3p (250 nM) alone. (K) Dependence of the actin-activated ATPase activity of three-step–purified Myo2 as a function of the concentrations of native (open circles) and recombinant (closed circles) GST-Rng3p. Conditions: 30 nM Myo2 and 10 μM actin filaments in 2 mM ATP, 3 mM MgCl 2 , 0.1 mM CaCl 2 , and 75 mM KCl. Error bars show SD. (L) Dependence of the number of actin filaments captured by two-step–purified Myo2 on the concentration of recombinant GST-Rng3p. All filaments in a 130 μm 2 frame of a fluorescence micrograph were counted. (squares) 20 nM two-step–purified Myo2. No gliding was observed in the absence of GST-Rng3p. (circles) 200 nM two-step–purified Myo2. These samples supported gliding in the absence of GST-Rng3p. (horizontal line) 150 nM crude Myo2 purified after overexpression from MLP 374 ( 3nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). These samples supported robust gliding.

Techniques Used: Activity Assay, Purification, SDS Page, Binding Assay, Affinity Purification, Positive Control, Negative Control, Recombinant, Staining, Fluorescence, Labeling, Flow Cytometry, Concentration Assay, Over Expression

Cdc4p and Rlc1p are the light chains for Myo2p. Quantitative analysis of GST-pull down experiments with GST-tagged myosin light chains, GST-tagged Myo2p heavy chain, or GFP-tagged candidate myosin light chains. Proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE, immunoblotted for Myo2p, Myo1p, or GFP, detected by ECL, and quantitated by densitometry of the bands. (A) TP 150 cells overexpressed GST-tagged candidate light chains cdc4 , rlc1 , cam1 , or cam2 . The GST-fusion proteins in cell extracts were affinity purified on glutathione-Sepharose and copurifying Myo2p (black bars) and Myo1p (gray bars) were quantitated by densitometry. Amounts were estimated relative to the maximal signal, which was given a value of 1. (B) Glutathione-Sepharose was used to purify GST-tagged proteins from cells expressing myo2-GST alone, YFP-cdc4 alone, rlc1-GFP alone, cam2-GFP alone, myo2-GST plu s YFP-cdc4 , myo2-GST plus rlc1-GFP , or myo2-GST plus cam2-GFP . Relative amounts of Myo2p (black bars) and copurifying light chains (gray bars) were quantitated by densitometry of bands detected after immunoblotting.
Figure Legend Snippet: Cdc4p and Rlc1p are the light chains for Myo2p. Quantitative analysis of GST-pull down experiments with GST-tagged myosin light chains, GST-tagged Myo2p heavy chain, or GFP-tagged candidate myosin light chains. Proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE, immunoblotted for Myo2p, Myo1p, or GFP, detected by ECL, and quantitated by densitometry of the bands. (A) TP 150 cells overexpressed GST-tagged candidate light chains cdc4 , rlc1 , cam1 , or cam2 . The GST-fusion proteins in cell extracts were affinity purified on glutathione-Sepharose and copurifying Myo2p (black bars) and Myo1p (gray bars) were quantitated by densitometry. Amounts were estimated relative to the maximal signal, which was given a value of 1. (B) Glutathione-Sepharose was used to purify GST-tagged proteins from cells expressing myo2-GST alone, YFP-cdc4 alone, rlc1-GFP alone, cam2-GFP alone, myo2-GST plu s YFP-cdc4 , myo2-GST plus rlc1-GFP , or myo2-GST plus cam2-GFP . Relative amounts of Myo2p (black bars) and copurifying light chains (gray bars) were quantitated by densitometry of bands detected after immunoblotting.

Techniques Used: SDS Page, Affinity Purification, Expressing

Effects of mutations in Myo2p light chains and Rng3p on motility activity. (A) Amino acid sequence alignment of the NH 2 -terminal regions of Rlc1p, Rlc1p-N1Δ, Rlc1p-N2Δ, D. melanogaster RLC, and Homo sapien RLC homologues. Alignment was generated with MacVector 7.1.1 and Boxshade software. Single (above) and double (below) lines on the alignment denote potential Rlc1p Ser/Thr phosphorylation sites and the phospho-regulatory Thr-Ser characteristic of higher eukaryotic RLCs, respectively. The box in Rlc1p-N2Δ indicates the additional amino acid substitutions. Black, amino acid identities; gray, amino acid similarities. (B) Viability of an rlc1 Δ strain (MLP 7) carrying empty vector (negative control), pGFP- rlc1 (positive control), pGFP- rlc1-N1 Δ, or pGFP- rlc1-N2 Δ. Transformants were streaked on an EMM Ura - plate containing 1M KCl. (C) Phenotypic quantitation of MLP 7 carrying empty vector (negative control), pGFP- rlc1 (positive control), or pGFP- rlc1-N1 Δ. Transformants were grown in liquid EMM Ura − media and their nuclei stained. Nuclei/cell were visualized and scored by fluorescence microscopy. (D) Localization of GFP-Rlc1p in MLP 7 containing either pGFP- rlc1 (left panels) or pGFP- rlc1-N1 Δ (right panels). Cells were grown in liquid EMM Ura − media. Fluorescence and DIC micrographs are shown. Bar, 5 μm. (E–I) Actin filament gliding assays using crude Myo2 (0.25 mg/ml impure protein) isolated in one step on glutathione-Sepharose from strains with mutations in Myo2p light chains and Rng3p. (E–G) Fluorescence micrographs of filaments labeled with rhodamine-phalloidin. Bars, 5 μm. (H and I) Quantitation of gliding rates. Conditions: samples were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Myo2 from a strain lacking Rlc1p (MLP 534 rlc1 Δ 41nmt1 promoter- myo2 plus pGST- cdc4 ). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Arrowheads mark the 6- s time point in filaments that moved. (F) Temperature dependence of actin filament attachment to crude, wild-type Myo2 (0.25 mg/ml) purified from MLP 509 ( 41nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). (G) Temperature dependence of actin filament attachment to crude Myo2 (0.25 mg/ml) purified from a strain with a temperature-sensitive mutation, rng3-65 (MLP 586 rng3-65 41nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). (H) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and rng3-65 (MLP 586) temperature-sensitive backgrounds. (I) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and cdc4 temperature-sensitive backgrounds. Strains: MLP 539 cdc4-8 41nmt1 promoter- myo2 , plus pGST- cdc4-8 and pGST- rlc1 ; MLP 641 cdc4-31 41nmt1 promoter- myo2 plus pGST- cdc4-31 and pGST- rlc1 ; MLP 647 cdc4-C2 41nmt1 promoter- myo2 plus pGST- cdc4-C2 and pGST- rlc1 ; MLP 648 cdc4-A2 41nmt1 promoter- myo2 plus pGST- cdc4-A2 and pGST- rlc1 .
Figure Legend Snippet: Effects of mutations in Myo2p light chains and Rng3p on motility activity. (A) Amino acid sequence alignment of the NH 2 -terminal regions of Rlc1p, Rlc1p-N1Δ, Rlc1p-N2Δ, D. melanogaster RLC, and Homo sapien RLC homologues. Alignment was generated with MacVector 7.1.1 and Boxshade software. Single (above) and double (below) lines on the alignment denote potential Rlc1p Ser/Thr phosphorylation sites and the phospho-regulatory Thr-Ser characteristic of higher eukaryotic RLCs, respectively. The box in Rlc1p-N2Δ indicates the additional amino acid substitutions. Black, amino acid identities; gray, amino acid similarities. (B) Viability of an rlc1 Δ strain (MLP 7) carrying empty vector (negative control), pGFP- rlc1 (positive control), pGFP- rlc1-N1 Δ, or pGFP- rlc1-N2 Δ. Transformants were streaked on an EMM Ura - plate containing 1M KCl. (C) Phenotypic quantitation of MLP 7 carrying empty vector (negative control), pGFP- rlc1 (positive control), or pGFP- rlc1-N1 Δ. Transformants were grown in liquid EMM Ura − media and their nuclei stained. Nuclei/cell were visualized and scored by fluorescence microscopy. (D) Localization of GFP-Rlc1p in MLP 7 containing either pGFP- rlc1 (left panels) or pGFP- rlc1-N1 Δ (right panels). Cells were grown in liquid EMM Ura − media. Fluorescence and DIC micrographs are shown. Bar, 5 μm. (E–I) Actin filament gliding assays using crude Myo2 (0.25 mg/ml impure protein) isolated in one step on glutathione-Sepharose from strains with mutations in Myo2p light chains and Rng3p. (E–G) Fluorescence micrographs of filaments labeled with rhodamine-phalloidin. Bars, 5 μm. (H and I) Quantitation of gliding rates. Conditions: samples were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Myo2 from a strain lacking Rlc1p (MLP 534 rlc1 Δ 41nmt1 promoter- myo2 plus pGST- cdc4 ). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Arrowheads mark the 6- s time point in filaments that moved. (F) Temperature dependence of actin filament attachment to crude, wild-type Myo2 (0.25 mg/ml) purified from MLP 509 ( 41nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). (G) Temperature dependence of actin filament attachment to crude Myo2 (0.25 mg/ml) purified from a strain with a temperature-sensitive mutation, rng3-65 (MLP 586 rng3-65 41nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). (H) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and rng3-65 (MLP 586) temperature-sensitive backgrounds. (I) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and cdc4 temperature-sensitive backgrounds. Strains: MLP 539 cdc4-8 41nmt1 promoter- myo2 , plus pGST- cdc4-8 and pGST- rlc1 ; MLP 641 cdc4-31 41nmt1 promoter- myo2 plus pGST- cdc4-31 and pGST- rlc1 ; MLP 647 cdc4-C2 41nmt1 promoter- myo2 plus pGST- cdc4-C2 and pGST- rlc1 ; MLP 648 cdc4-A2 41nmt1 promoter- myo2 plus pGST- cdc4-A2 and pGST- rlc1 .

Techniques Used: Activity Assay, Sequencing, Generated, Software, Plasmid Preparation, Negative Control, Positive Control, Quantitation Assay, Staining, Fluorescence, Microscopy, Isolation, Labeling, Flow Cytometry, Purification, Mutagenesis, In Vitro

13) Product Images from "The TRPM7 chanzyme is cleaved to release a chromatin modifying kinase"

Article Title: The TRPM7 chanzyme is cleaved to release a chromatin modifying kinase

Journal: Cell

doi: 10.1016/j.cell.2014.03.046

TRPM7 binds transcription factors and subunits of chromatin remodeling complexes A. Endogenous TRPM7 was IP’d from combined cytosolic+nuclear extracts of WT3 or KO9 mESC with αA25 mouse or αC47 rabbit antibody and the IP probed on WB with antibody to the indicated proteins. INO80 cell lysate lanes were run on a different gel, as indicated by gap. B. Subunits of Polycomb and INO80 chromatin remodeling complexes bind ectopically expressed M7CKs. FLAG-tagged transcription factors were transiently co-expressed in 293T cells with HA-tagged M7CK-L, -M or -S. Cytosolic+nuclear extract was IP’d with αHA-agarose and co-IP’d proteins were probed on WB with αFLAG-peroxidase. C. Affinity-purified long form of M7CK (M7CK-L) binds purified transcription factors (Coomassie). Left 3 panels : HA-tagged M7CK-L (M7) was combined with purified YY1 or Ezh2 protein complexes and IP’d with αHA-agarose. Right panel : purified GST or GST-RYBP fragment (M7CK-binding domain, a. a. 22–47, GST-RYBP bd ) fusion proteins were combined with purified M7CK-L and GST pulled down with glutathione agarose.
Figure Legend Snippet: TRPM7 binds transcription factors and subunits of chromatin remodeling complexes A. Endogenous TRPM7 was IP’d from combined cytosolic+nuclear extracts of WT3 or KO9 mESC with αA25 mouse or αC47 rabbit antibody and the IP probed on WB with antibody to the indicated proteins. INO80 cell lysate lanes were run on a different gel, as indicated by gap. B. Subunits of Polycomb and INO80 chromatin remodeling complexes bind ectopically expressed M7CKs. FLAG-tagged transcription factors were transiently co-expressed in 293T cells with HA-tagged M7CK-L, -M or -S. Cytosolic+nuclear extract was IP’d with αHA-agarose and co-IP’d proteins were probed on WB with αFLAG-peroxidase. C. Affinity-purified long form of M7CK (M7CK-L) binds purified transcription factors (Coomassie). Left 3 panels : HA-tagged M7CK-L (M7) was combined with purified YY1 or Ezh2 protein complexes and IP’d with αHA-agarose. Right panel : purified GST or GST-RYBP fragment (M7CK-binding domain, a. a. 22–47, GST-RYBP bd ) fusion proteins were combined with purified M7CK-L and GST pulled down with glutathione agarose.

Techniques Used: Western Blot, Co-Immunoprecipitation Assay, Affinity Purification, Purification, Binding Assay

Zinc dependence of M7CK binding to zinc-finger domain (ZfD)-containing proteins A. HA-tagged M7CK was co-expressed with FLAG-tagged interacting proteins in 293T cells. The cell lysate was split into equal aliquots and supplemented with 20 μM TPEN and 50 μM of the indicated ions. M7CK was IP’d with αHA and the co-IP’d proteins probed with αFLAG. B. FLAG-tagged ZfD-containing proteins were co-expressed in 293T cells with HA-tagged M7CK. [Zn 2+ ] was adjusted by variation of [EGTA] and [ZnSO 4 ]. M7CK-HA was IP’d with HA-agarose; ZfD protein binding was detected in the αFLAG WB. C [Zn 2+ ] dependence of M7CK binding to GST-RYBP ZfD. Purified GST fusion with RYBP fragment (a. a. 22–47 comprising the RYBP-ZfD) was combined with purified M7CK-L in the presence of varying [Zn 2+ ] and pulled down by glutathione agarose (Coomassie-stained gel). D. Quantification of M7CK binding shown in C . Values of integrated pixels of the bound M7CK image were normalized to maximum binding at 10 μM [Zn 2+ ]. Fitted equation and parameters shown.
Figure Legend Snippet: Zinc dependence of M7CK binding to zinc-finger domain (ZfD)-containing proteins A. HA-tagged M7CK was co-expressed with FLAG-tagged interacting proteins in 293T cells. The cell lysate was split into equal aliquots and supplemented with 20 μM TPEN and 50 μM of the indicated ions. M7CK was IP’d with αHA and the co-IP’d proteins probed with αFLAG. B. FLAG-tagged ZfD-containing proteins were co-expressed in 293T cells with HA-tagged M7CK. [Zn 2+ ] was adjusted by variation of [EGTA] and [ZnSO 4 ]. M7CK-HA was IP’d with HA-agarose; ZfD protein binding was detected in the αFLAG WB. C [Zn 2+ ] dependence of M7CK binding to GST-RYBP ZfD. Purified GST fusion with RYBP fragment (a. a. 22–47 comprising the RYBP-ZfD) was combined with purified M7CK-L in the presence of varying [Zn 2+ ] and pulled down by glutathione agarose (Coomassie-stained gel). D. Quantification of M7CK binding shown in C . Values of integrated pixels of the bound M7CK image were normalized to maximum binding at 10 μM [Zn 2+ ]. Fitted equation and parameters shown.

Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Protein Binding, Western Blot, Purification, Staining

TRPM7 cleavage fragments identified in multiple cell lines and tissues A. TRPM7 protein cleavage fragments in mouse mesangial SV40 mes13 cells. Cells were extracted with TBS/1% NP40. Endogenous TRPM7 was immunoprecipitated (IP’d) from lysates with TRPM7 C-terminal mouse monoclonal antibody (αA25) or normal mouse IgG and probed on WB with anti-C-terminal rabbit antibody (αC47). C-terminally HA-tagged TRPM7 (expressed=expr) was IP’d with anti-HA-agarose (αHA) from SV40 mes13 cells stably expressing recombinant protein and probed on WB with αHA-peroxidase conjugate. Scale ( left ) indicates the molecular weight of major bands calculated from their electrophoretic mobility relative to standard molecular weight markers. Cartoon ( right ) shows the approximate position of cleavage sites; K indicates kinase domain. B. TRPM7 cleavage pattern in 8 distinct cell lines. Mouse mesangial (SV40 mes13), macrophage (RAW 264.7), mESC, human B-lymphocyte (Raji), Caco-2 (colon epithelial), prostate (metastatic LNCaP and non-metastatic RWPE1), and embryonic kidney (HEK-293) cells were extracted and IP’d as described in A . Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from each tissue. Information about the relative content of the full length TRPM7 and the cleaved fragments is contained in each individual lane, which are intentionally not normalized to control protein. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (not shown). mESCs were generated as described in Experimental Procedures from WT or TrpM7 −/− (KO) blastocysts. The lower panel in the mESC column shows equal actin content in both mESC lysates. Samples run on different gels are combined in the figure and aligned against identical molecular weight markers. C. TRPM7 cleavage pattern in different mouse tissues. Freshly isolated mouse organs were extracted and IP’d as described in A . Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from in each tissue. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (not shown).
Figure Legend Snippet: TRPM7 cleavage fragments identified in multiple cell lines and tissues A. TRPM7 protein cleavage fragments in mouse mesangial SV40 mes13 cells. Cells were extracted with TBS/1% NP40. Endogenous TRPM7 was immunoprecipitated (IP’d) from lysates with TRPM7 C-terminal mouse monoclonal antibody (αA25) or normal mouse IgG and probed on WB with anti-C-terminal rabbit antibody (αC47). C-terminally HA-tagged TRPM7 (expressed=expr) was IP’d with anti-HA-agarose (αHA) from SV40 mes13 cells stably expressing recombinant protein and probed on WB with αHA-peroxidase conjugate. Scale ( left ) indicates the molecular weight of major bands calculated from their electrophoretic mobility relative to standard molecular weight markers. Cartoon ( right ) shows the approximate position of cleavage sites; K indicates kinase domain. B. TRPM7 cleavage pattern in 8 distinct cell lines. Mouse mesangial (SV40 mes13), macrophage (RAW 264.7), mESC, human B-lymphocyte (Raji), Caco-2 (colon epithelial), prostate (metastatic LNCaP and non-metastatic RWPE1), and embryonic kidney (HEK-293) cells were extracted and IP’d as described in A . Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from each tissue. Information about the relative content of the full length TRPM7 and the cleaved fragments is contained in each individual lane, which are intentionally not normalized to control protein. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (not shown). mESCs were generated as described in Experimental Procedures from WT or TrpM7 −/− (KO) blastocysts. The lower panel in the mESC column shows equal actin content in both mESC lysates. Samples run on different gels are combined in the figure and aligned against identical molecular weight markers. C. TRPM7 cleavage pattern in different mouse tissues. Freshly isolated mouse organs were extracted and IP’d as described in A . Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from in each tissue. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (not shown).

Techniques Used: Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Recombinant, Molecular Weight, Isolation, Generated

14) Product Images from "Phosphorylation of E-cadherin at threonine 790 by protein kinase Cδ reduces β-catenin binding and suppresses the function of E-cadherin"

Article Title: Phosphorylation of E-cadherin at threonine 790 by protein kinase Cδ reduces β-catenin binding and suppresses the function of E-cadherin

Journal: Oncotarget

doi: 10.18632/oncotarget.9403

Phosphorylation of E-cadherin at Thr-790 diminishes its interaction with β-catenin A. The diagram depicts the regions of E-cadherin for p120-catenin and β-catenin binding. Note that Thr790 resides in the β-catenin binding region. B. Simulation for the interface between E-cadherin and β-catenin. (a) The structure of the E-cadherin/β-catenin complex [Protein Data Bank (PDB) ID code: 1I7X]. E-cadherin (a.a 782-838) is represented with a red tube and β-catenin is shown as a surface representation (light grey). The T790 residue of E-cadherin is represented by a yellow stick and β-catenin N430 is represented by green space filling. (b) A close-up view of the interaction of β-catenin N430 (represented by a green stick) with E-cadherin T790 (represented by a red stick). Hydrogen bonds are shown as blue dashed lines. (c) The side chain of pT790 (shown in space filling representation) clashes with β-catenin (shown in surface representation). C. CHO cells stably expressing E-cadherin wt or T790E were grown to confluence and were then stained with anti-β-catenin and anti-E-cadherin (ECCD-2). Note that β-catenin is less organized at the cell-cell contacts of the CHO cells expressing E-cadherin T790E. D. CHO cells, as in Figure 4A , were grown to confluence and then were subjected to cell surface biotinylation. For measurement of the membrane bound level of β-catenin, equal amounts of cell lysates were incubated with avidin-immobilized agarose beads and the complexes were analyzed by immunoblotting with anti-β-catenin. For measurement of the total expression level of β-catenin, equal amounts of cell lysates were analyzed by immunoblotting with anti-β-catenin. E. MDCK cells stably expressing GFP, GFP-PKC (wt) or (kd) mutant were grown to confluence and then lysed. Equal amounts of cell lysates were immunoprecipitated by anti-E-cadherin antibody (clone 36) and the immunocomplexes were analyzed by immunoblotting with antibodies to β-catenin and E-cadherin. The whole cell lysates were analyzed by immunoblotting with the indicated antibodies. F. MDCK cells were treated with (+) or without (−) HGF (20 ng/ml) for 1 hr. Equal amounts of cell lysates were immunoprecipitated by anti-E-cadherin antibody (clone 36) and the immunocomplexes were analyzed by immunoblotting with antibodies to β-catenin and E-cadherin. The whole cell lysates were analyzed by immunoblotting with the indicated antibodies. G. Purified β-catenin was incubated with purified GST-E-cadherin-cytoplasmic domain (cd) or GST as a control. The protein complexes were pulled-down by glutathione agarose beads. After washing, the protein complexes were analyzed by immunoblotting with anti-β-catenin. The level of bound β-catenin was quantified and expressed as the fold relative to the level in the GST-E-cadherin-cd wt. The values (mean ± SD) are from three experiments. *, P
Figure Legend Snippet: Phosphorylation of E-cadherin at Thr-790 diminishes its interaction with β-catenin A. The diagram depicts the regions of E-cadherin for p120-catenin and β-catenin binding. Note that Thr790 resides in the β-catenin binding region. B. Simulation for the interface between E-cadherin and β-catenin. (a) The structure of the E-cadherin/β-catenin complex [Protein Data Bank (PDB) ID code: 1I7X]. E-cadherin (a.a 782-838) is represented with a red tube and β-catenin is shown as a surface representation (light grey). The T790 residue of E-cadherin is represented by a yellow stick and β-catenin N430 is represented by green space filling. (b) A close-up view of the interaction of β-catenin N430 (represented by a green stick) with E-cadherin T790 (represented by a red stick). Hydrogen bonds are shown as blue dashed lines. (c) The side chain of pT790 (shown in space filling representation) clashes with β-catenin (shown in surface representation). C. CHO cells stably expressing E-cadherin wt or T790E were grown to confluence and were then stained with anti-β-catenin and anti-E-cadherin (ECCD-2). Note that β-catenin is less organized at the cell-cell contacts of the CHO cells expressing E-cadherin T790E. D. CHO cells, as in Figure 4A , were grown to confluence and then were subjected to cell surface biotinylation. For measurement of the membrane bound level of β-catenin, equal amounts of cell lysates were incubated with avidin-immobilized agarose beads and the complexes were analyzed by immunoblotting with anti-β-catenin. For measurement of the total expression level of β-catenin, equal amounts of cell lysates were analyzed by immunoblotting with anti-β-catenin. E. MDCK cells stably expressing GFP, GFP-PKC (wt) or (kd) mutant were grown to confluence and then lysed. Equal amounts of cell lysates were immunoprecipitated by anti-E-cadherin antibody (clone 36) and the immunocomplexes were analyzed by immunoblotting with antibodies to β-catenin and E-cadherin. The whole cell lysates were analyzed by immunoblotting with the indicated antibodies. F. MDCK cells were treated with (+) or without (−) HGF (20 ng/ml) for 1 hr. Equal amounts of cell lysates were immunoprecipitated by anti-E-cadherin antibody (clone 36) and the immunocomplexes were analyzed by immunoblotting with antibodies to β-catenin and E-cadherin. The whole cell lysates were analyzed by immunoblotting with the indicated antibodies. G. Purified β-catenin was incubated with purified GST-E-cadherin-cytoplasmic domain (cd) or GST as a control. The protein complexes were pulled-down by glutathione agarose beads. After washing, the protein complexes were analyzed by immunoblotting with anti-β-catenin. The level of bound β-catenin was quantified and expressed as the fold relative to the level in the GST-E-cadherin-cd wt. The values (mean ± SD) are from three experiments. *, P

Techniques Used: Binding Assay, Stable Transfection, Expressing, Staining, Incubation, Avidin-Biotin Assay, Mutagenesis, Immunoprecipitation, Purification

Phosphorylation of E-cadherin at Thr-790 by PKCδ impairs the homophilic interaction of E-cadherin A. CHO cells stably expressing E-cadherin or its mutants (T790E and T790A) and their neomycin-resistant control cells (neo) were established by a lentiviral expression system. Those cells were subjected to biotinylation with sulfo-NHS-biotin. For measurement of the cell surface level of E-cadherin, equal amounts of cell lysates were incubated with avidin-immobilized agarose beads and then the complexes were analyzed by immunoblotting with anti-E-cadherin. For measurement of the total expression level of E-cadherin, equal amounts of cell lysates were analyzed by immunoblotting with anti-E-cadherin (clone 36). B. CHO cells, as in panel (A), were grown to confluence and then lysed. E-cadherin was immunoprecipitated by anti-E-cadherin (clone 36) and the immunocomplexes were analyzed by immunoblotting with antibodies to E-cadherin and E-cadherin pT790. C. CHO cells, as in panel (A), were collected by trypsinization, suspended in medium with 10% serum, and subjected to a constant rotation at 0.5 xg. Two days later, the number of cell aggregates 400 μm or larger in diameter was measured under a phase-contrast microscope. The values (mean ± SD) are from three experiments. *, P
Figure Legend Snippet: Phosphorylation of E-cadherin at Thr-790 by PKCδ impairs the homophilic interaction of E-cadherin A. CHO cells stably expressing E-cadherin or its mutants (T790E and T790A) and their neomycin-resistant control cells (neo) were established by a lentiviral expression system. Those cells were subjected to biotinylation with sulfo-NHS-biotin. For measurement of the cell surface level of E-cadherin, equal amounts of cell lysates were incubated with avidin-immobilized agarose beads and then the complexes were analyzed by immunoblotting with anti-E-cadherin. For measurement of the total expression level of E-cadherin, equal amounts of cell lysates were analyzed by immunoblotting with anti-E-cadherin (clone 36). B. CHO cells, as in panel (A), were grown to confluence and then lysed. E-cadherin was immunoprecipitated by anti-E-cadherin (clone 36) and the immunocomplexes were analyzed by immunoblotting with antibodies to E-cadherin and E-cadherin pT790. C. CHO cells, as in panel (A), were collected by trypsinization, suspended in medium with 10% serum, and subjected to a constant rotation at 0.5 xg. Two days later, the number of cell aggregates 400 μm or larger in diameter was measured under a phase-contrast microscope. The values (mean ± SD) are from three experiments. *, P

Techniques Used: Stable Transfection, Expressing, Incubation, Avidin-Biotin Assay, Immunoprecipitation, Microscopy

The expression of PKCδ is correlated with Thr790 phosphorylation of E-cadherin in cervical carcinoma A. Two subclones (#1 and #2) of human cervical carcinoma CaSki cells were selected on the basis of the PKCδ expression level. The cell lysates were analyzed by immunoblotting with the indicated antibodies. The level of E-cadherin pT790 and PKCδ were quantified and expressed as fold relative to the level in the CaSki #1 cells. B. The cells, as in panel (A), were grown to confluence and were then stained with anti-E-cadherin antibodies (clone 36 and ECCD-2). The profiles of the E-cadherin fluorescence intensity (F. I.; a.u. arbitrary units) along the white lines were shown with line graphs. The scale bar represents 10 μm. C. Two subclones (#1 and #2) of CaSki cells were grown to confluence and then subjected to cell surface biotinylation. For measurement of the membrane bound level of β-catenin, equal amounts of cell lysates were incubated with avidin-immobilized agarose beads and the complexes were analyzed by immunoblotting with anti-β-catenin and anti-E-cadherin. For measurement of the total expression level of E-cadherin and β-catenin, equal amounts of cell lysates were analyzed by immunoblotting with anti-E-cadherin and anti-β-catenin. D. CaSki #1 cells were treated with (+) or without (−) HGF (30 ng/ml) for 15 min. Equal amounts of cell lysates were immunoprecipitated by anti-E-cadherin antibody (clone 36) and the immunocomplexes were analyzed by immunoblotting with antibodies to β-catenin and E-cadherin. The level of E-cadherin pT790, PKCδ pY311 and the β-catenin were measured and expressed as the fold relative to the level of the control cells. E. Two subclones (#1 and #2) of CaSki cells were allowed to grow as colonies and treated with or without HGF for 12 h. The percentage of scattered colonies out of the total counted cell colonies (n ≥ 100) was determined. The values (mean ± SD) are from three experiments. **, P
Figure Legend Snippet: The expression of PKCδ is correlated with Thr790 phosphorylation of E-cadherin in cervical carcinoma A. Two subclones (#1 and #2) of human cervical carcinoma CaSki cells were selected on the basis of the PKCδ expression level. The cell lysates were analyzed by immunoblotting with the indicated antibodies. The level of E-cadherin pT790 and PKCδ were quantified and expressed as fold relative to the level in the CaSki #1 cells. B. The cells, as in panel (A), were grown to confluence and were then stained with anti-E-cadherin antibodies (clone 36 and ECCD-2). The profiles of the E-cadherin fluorescence intensity (F. I.; a.u. arbitrary units) along the white lines were shown with line graphs. The scale bar represents 10 μm. C. Two subclones (#1 and #2) of CaSki cells were grown to confluence and then subjected to cell surface biotinylation. For measurement of the membrane bound level of β-catenin, equal amounts of cell lysates were incubated with avidin-immobilized agarose beads and the complexes were analyzed by immunoblotting with anti-β-catenin and anti-E-cadherin. For measurement of the total expression level of E-cadherin and β-catenin, equal amounts of cell lysates were analyzed by immunoblotting with anti-E-cadherin and anti-β-catenin. D. CaSki #1 cells were treated with (+) or without (−) HGF (30 ng/ml) for 15 min. Equal amounts of cell lysates were immunoprecipitated by anti-E-cadherin antibody (clone 36) and the immunocomplexes were analyzed by immunoblotting with antibodies to β-catenin and E-cadherin. The level of E-cadherin pT790, PKCδ pY311 and the β-catenin were measured and expressed as the fold relative to the level of the control cells. E. Two subclones (#1 and #2) of CaSki cells were allowed to grow as colonies and treated with or without HGF for 12 h. The percentage of scattered colonies out of the total counted cell colonies (n ≥ 100) was determined. The values (mean ± SD) are from three experiments. **, P

Techniques Used: Expressing, Staining, Fluorescence, Incubation, Avidin-Biotin Assay, Immunoprecipitation

15) Product Images from "14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival *14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival * S⃞"

Article Title: 14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival *14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M807637200

The 14-3-3ζ-T179F mutant is able to bind phosphoserine/threonine target proteins and heterodimerize with other isoforms of 14-3-3. HEK 293T cells were lysed and whole cell lysates subjected to pulldown experiments using either GST-Sepharose, GST-14-3-3ζ-Sepharose, or GST-14-3-3ζ T179F-Sepharose. Pulldowns were examined by immunoblot analysis using anti-Raf-1 and anti-BAD pAb ( A ) or a phospho-specific antibody that recognizes phosphorylated 14-3-3 binding motifs (Cell Signaling) ( B ). C , recombinant purified 14-3-3ζ was phosphorylated with c-Src (Tyr(P) 179 -14-3-3ζ) or left nonphosphorylated ( non-phospho-14-3-3 ζ) and then incubated with either GST-SH2 Shc or GST alone bound to glutathione-Sepharose resin. The resin was washed to remove unbound 14-3-3ζ, and the assembled complexes (the complex assembled in lane 1 and used in the pulldown experiment is illustrated) were then used to pulldown 14-3-3-binding proteins from HEK 293T cell lysates. The pulldowns were washed in Nonidet P-40 lysis buffer and subjected to immunoblot analysis with phospho-specific antibodies that recognize phosphorylated 14-3-3 binding motifs or anti-Raf-1 pAb. D , HEK-293T cells were either mock transfected or transfected with constructs for the expression of wt 14-3-3ζ-Myc or the T179F mutant. After 48 h, the cells were lysed in Nonidet P-40 lysis buffer and subjected to immunoprecipitation using the 9E10 mAb. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblot analysis with phospho-specific antibodies that recognize phosphorylated 14-3-3 binding motifs ( top panel ) or the 9E10 mAb ( bottom panel ). E , HEK-293T cells were co-transfected with the indicated combinations of 14-3-3 isoforms. After 48 h, the cells were lysed, and lysates were immunoprecipitated ( IP ) with the 9E10 mAb. Immunoprecipitates and whole cell lysates ( WCL ) were subjected to SDS-PAGE and immunoblotted using the 9E10 mAb or the 12CA5 mAb. The 14-3-3ζ-Myc-T179F mutant was able to co-immunoprecipitate with 14-3-3γ-EE (data not shown), 14-3-3ζ-HA, and 14-3-3τ-HA. WB , Western blotting.
Figure Legend Snippet: The 14-3-3ζ-T179F mutant is able to bind phosphoserine/threonine target proteins and heterodimerize with other isoforms of 14-3-3. HEK 293T cells were lysed and whole cell lysates subjected to pulldown experiments using either GST-Sepharose, GST-14-3-3ζ-Sepharose, or GST-14-3-3ζ T179F-Sepharose. Pulldowns were examined by immunoblot analysis using anti-Raf-1 and anti-BAD pAb ( A ) or a phospho-specific antibody that recognizes phosphorylated 14-3-3 binding motifs (Cell Signaling) ( B ). C , recombinant purified 14-3-3ζ was phosphorylated with c-Src (Tyr(P) 179 -14-3-3ζ) or left nonphosphorylated ( non-phospho-14-3-3 ζ) and then incubated with either GST-SH2 Shc or GST alone bound to glutathione-Sepharose resin. The resin was washed to remove unbound 14-3-3ζ, and the assembled complexes (the complex assembled in lane 1 and used in the pulldown experiment is illustrated) were then used to pulldown 14-3-3-binding proteins from HEK 293T cell lysates. The pulldowns were washed in Nonidet P-40 lysis buffer and subjected to immunoblot analysis with phospho-specific antibodies that recognize phosphorylated 14-3-3 binding motifs or anti-Raf-1 pAb. D , HEK-293T cells were either mock transfected or transfected with constructs for the expression of wt 14-3-3ζ-Myc or the T179F mutant. After 48 h, the cells were lysed in Nonidet P-40 lysis buffer and subjected to immunoprecipitation using the 9E10 mAb. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblot analysis with phospho-specific antibodies that recognize phosphorylated 14-3-3 binding motifs ( top panel ) or the 9E10 mAb ( bottom panel ). E , HEK-293T cells were co-transfected with the indicated combinations of 14-3-3 isoforms. After 48 h, the cells were lysed, and lysates were immunoprecipitated ( IP ) with the 9E10 mAb. Immunoprecipitates and whole cell lysates ( WCL ) were subjected to SDS-PAGE and immunoblotted using the 9E10 mAb or the 12CA5 mAb. The 14-3-3ζ-Myc-T179F mutant was able to co-immunoprecipitate with 14-3-3γ-EE (data not shown), 14-3-3ζ-HA, and 14-3-3τ-HA. WB , Western blotting.

Techniques Used: Mutagenesis, Binding Assay, Recombinant, Purification, Incubation, Lysis, Transfection, Construct, Expressing, Immunoprecipitation, SDS Page, Western Blot

14-3-3ζ can simultaneously bind phosphoserine residues and Shc. CTL-EN cells were electroporated with either wt 14-3-3ζ-Myc or the T179F mutant following which the cells were factor-deprived overnight and then stimulated with 50 ng/ml GM-CSF for 5 min. The cells were then lysed and pulldowns ( PD ) were performed using either a Ser(P) 585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG) or a non-phospho-Ser 585 control peptide (Biotin-NHS-KGGFDFNGPYLGPPHSRSLPDGG) adsorbed to streptavidin-Sepharose. PD and whole cell lysates ( WCL ) were subjected to SDS-PAGE and immunoblot analysis using anti-Shc pAb and the 9E10 mAb. The results are typical of two experiments.
Figure Legend Snippet: 14-3-3ζ can simultaneously bind phosphoserine residues and Shc. CTL-EN cells were electroporated with either wt 14-3-3ζ-Myc or the T179F mutant following which the cells were factor-deprived overnight and then stimulated with 50 ng/ml GM-CSF for 5 min. The cells were then lysed and pulldowns ( PD ) were performed using either a Ser(P) 585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG) or a non-phospho-Ser 585 control peptide (Biotin-NHS-KGGFDFNGPYLGPPHSRSLPDGG) adsorbed to streptavidin-Sepharose. PD and whole cell lysates ( WCL ) were subjected to SDS-PAGE and immunoblot analysis using anti-Shc pAb and the 9E10 mAb. The results are typical of two experiments.

Techniques Used: CTL Assay, Mutagenesis, SDS Page

14-3-3 proteins undergo tyrosine phosphorylation in response to GM-CSF stimulation. A , primary human mononuclear cells from peripheral blood ( left panel ) or CTL-EN cells ( right panel ) were stimulated with GM-CSF. Endogenous 14-3-3 was precipitated with a Ser(P) 585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG; to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-Shc and anti-14-3-3 pAbs. B , factor-dependent CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight in medium containing 0.5% FCS and then stimulated with GM-CSF. The cells were lysed, and pulldown experiments were performed using either GST-SH2 Shc (to precipitate tyrosine-phosphorylated endogenous 14-3-3) or the Ser(P) 585 peptide (to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-14-3-3 pAb. C , HEK 293T cells were transfected with a construct for the expression of wt 14-3-3ζ-Myc. After 48 h, the cells were lysed and 14-3-3ζ-Myc immunoprecipitated using the 9E10 mAb. Immunoprecipitates were subjected to immunoblot analysis with either the 4G10 mAb (Millipore) ( top panel ), an anti-phosphotyrosine pAb (Biomol) ( middle panel ) or the 9E10 mAb ( bottom panel ). D ). The location of tyrosines 19, 118, 178, 179, and 211 of 14-3-3ζ are indicated. E and F , recombinant purified 14-3-3ζ and 14-3-3ζ-T179F were phosphorylated in vitro using c-Src and then subjected to immunoblot analysis using the 4G10 anti-phosphotyrosine mAb ( anti-PY ) ( E, top panel ), anti-Tyr(P) 179 -14-3-3ζ pAb ( anti-PY179 ) ( F, top panel ) or anti-14-3-3ζ pAb to indicate loading ( E and F, bottom panels ). G , HEK-293T cells were transfected with constructs for the expression of wt 14-3-3ζ-Myc or the T179F mutant. After 48 h, the cells were stimulated for 15 min with sodium pervanadate (VO 4 ) (+) or left unstimulated (-). The cells were then lysed and subjected to immunoprecipitation using the 9E10 mAb. Immunoprecipitated proteins were subjected to immunoblot analysis using the anti-Tyr(P) 179 -14-3-3ζ pAb ( G, top panel ) and the 9E10 mAb ( G, bottom panel ). H , CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight. The cells were then stimulated with 50 ng/ml GM-CSF or sodium pervanadate (VO 4 ), following which the cells were lysed, and endogenous 14-3-3 was precipitated ( PD ) using either a Ser(P) 585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG) or an Ser 585 (non-phospho-Ser 585 ) control peptide (Biotin-NHS-KGGFDFNGPYLGPPHSRSLPDGG) adsorbed to streptavidin-Sepharose resin. Pulldowns were subjected to immunoblot analysis using the anti-Tyr(P) 179 -14-3-3ζ pAb or anti-14-3-3 pAb. I , primary human mononuclear cells were stimulated with GM-CSF and total endogenous 14-3-3 was precipitated as in A . Precipitates were blotted with anti-Tyr(P) 179 -14-3-3ζ pAb or anti-14-3-3 pAb. J , the UT7 factor-dependent cell line was factor-deprived overnight and then stimulated with 50 ng/ml GM-CSF, following which the cells were lysed, and the βc subunit of the GM-CSF receptor was immunoprecipitated using the 8E4 and 1C1 mAbs. Immunoprecipitates were then subjected to immunoblot analysis with either anti-Tyr(P) 179 pAb ( top panel ) or 1C1 anti-βc mAb ( bottom panel ). The results are typical of at least two experiments. WB , Western blotting.
Figure Legend Snippet: 14-3-3 proteins undergo tyrosine phosphorylation in response to GM-CSF stimulation. A , primary human mononuclear cells from peripheral blood ( left panel ) or CTL-EN cells ( right panel ) were stimulated with GM-CSF. Endogenous 14-3-3 was precipitated with a Ser(P) 585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG; to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-Shc and anti-14-3-3 pAbs. B , factor-dependent CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight in medium containing 0.5% FCS and then stimulated with GM-CSF. The cells were lysed, and pulldown experiments were performed using either GST-SH2 Shc (to precipitate tyrosine-phosphorylated endogenous 14-3-3) or the Ser(P) 585 peptide (to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-14-3-3 pAb. C , HEK 293T cells were transfected with a construct for the expression of wt 14-3-3ζ-Myc. After 48 h, the cells were lysed and 14-3-3ζ-Myc immunoprecipitated using the 9E10 mAb. Immunoprecipitates were subjected to immunoblot analysis with either the 4G10 mAb (Millipore) ( top panel ), an anti-phosphotyrosine pAb (Biomol) ( middle panel ) or the 9E10 mAb ( bottom panel ). D ). The location of tyrosines 19, 118, 178, 179, and 211 of 14-3-3ζ are indicated. E and F , recombinant purified 14-3-3ζ and 14-3-3ζ-T179F were phosphorylated in vitro using c-Src and then subjected to immunoblot analysis using the 4G10 anti-phosphotyrosine mAb ( anti-PY ) ( E, top panel ), anti-Tyr(P) 179 -14-3-3ζ pAb ( anti-PY179 ) ( F, top panel ) or anti-14-3-3ζ pAb to indicate loading ( E and F, bottom panels ). G , HEK-293T cells were transfected with constructs for the expression of wt 14-3-3ζ-Myc or the T179F mutant. After 48 h, the cells were stimulated for 15 min with sodium pervanadate (VO 4 ) (+) or left unstimulated (-). The cells were then lysed and subjected to immunoprecipitation using the 9E10 mAb. Immunoprecipitated proteins were subjected to immunoblot analysis using the anti-Tyr(P) 179 -14-3-3ζ pAb ( G, top panel ) and the 9E10 mAb ( G, bottom panel ). H , CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight. The cells were then stimulated with 50 ng/ml GM-CSF or sodium pervanadate (VO 4 ), following which the cells were lysed, and endogenous 14-3-3 was precipitated ( PD ) using either a Ser(P) 585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG) or an Ser 585 (non-phospho-Ser 585 ) control peptide (Biotin-NHS-KGGFDFNGPYLGPPHSRSLPDGG) adsorbed to streptavidin-Sepharose resin. Pulldowns were subjected to immunoblot analysis using the anti-Tyr(P) 179 -14-3-3ζ pAb or anti-14-3-3 pAb. I , primary human mononuclear cells were stimulated with GM-CSF and total endogenous 14-3-3 was precipitated as in A . Precipitates were blotted with anti-Tyr(P) 179 -14-3-3ζ pAb or anti-14-3-3 pAb. J , the UT7 factor-dependent cell line was factor-deprived overnight and then stimulated with 50 ng/ml GM-CSF, following which the cells were lysed, and the βc subunit of the GM-CSF receptor was immunoprecipitated using the 8E4 and 1C1 mAbs. Immunoprecipitates were then subjected to immunoblot analysis with either anti-Tyr(P) 179 pAb ( top panel ) or 1C1 anti-βc mAb ( bottom panel ). The results are typical of at least two experiments. WB , Western blotting.

Techniques Used: CTL Assay, Expressing, Transfection, Construct, Immunoprecipitation, Recombinant, Purification, In Vitro, Mutagenesis, Western Blot

16) Product Images from "Modulation of Histone Deposition by the Karyopherin Kap114"

Article Title: Modulation of Histone Deposition by the Karyopherin Kap114

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.5.1764-1778.2005

Characterization of Nap1p point mutants. (a) Alanine mutations of Nap1p abolish its Kap114p binding. Binding assays were performed on glutathione-Sepharose with 2 μM GST or a 400 nM concentration of each GST-Nap1p mutant in the presence of 300 nM MBP-Kap114p. Eighty percent of the bound material was analyzed by SDS-PAGE and Coomassie blue (CBB) staining. The remaining material was analyzed by Western blotting (WB) with an anti-MBP antibody. The schematic shows the relative positions of alanine mutations in the Kap114p-binding domain (BD)of Nap1p. (b) Plasmid supercoiling assays were performed with 500 nM wild-type Nap1p or 250 and 500 nM concentrations of the indicated Nap1p mutants. (c) A Kap114-PrA-expressing Δnap1 strain was constructed and transformed with vector alone or with a plasmid expressing wild-type Nap1p or the indicated Nap1p mutant. The cytosol was prepared from each strain and analyzed for the expression of Nap1p and Kap114-PrA by Western blotting (input). The same cytosol was used for immunoisolation of Kap114-PrA and associated proteins (IP). After being washed, the associated proteins were eluted with a buffer containing the indicated concentrations of MgCl 2 and then analyzed by Western blotting for Nap1p and Kap114-PrA. (d) Binding between MBP-Kap114p and the indicated GST proteins was analyzed as described for panel a in the presence or absence of core histones (500 nM) and RanQ69L (20 μM), as indicated. (e and f) The localization of the indicated GFP-tagged proteins was analyzed in wild-type yeast. Coincident Hoechst staining is also shown.
Figure Legend Snippet: Characterization of Nap1p point mutants. (a) Alanine mutations of Nap1p abolish its Kap114p binding. Binding assays were performed on glutathione-Sepharose with 2 μM GST or a 400 nM concentration of each GST-Nap1p mutant in the presence of 300 nM MBP-Kap114p. Eighty percent of the bound material was analyzed by SDS-PAGE and Coomassie blue (CBB) staining. The remaining material was analyzed by Western blotting (WB) with an anti-MBP antibody. The schematic shows the relative positions of alanine mutations in the Kap114p-binding domain (BD)of Nap1p. (b) Plasmid supercoiling assays were performed with 500 nM wild-type Nap1p or 250 and 500 nM concentrations of the indicated Nap1p mutants. (c) A Kap114-PrA-expressing Δnap1 strain was constructed and transformed with vector alone or with a plasmid expressing wild-type Nap1p or the indicated Nap1p mutant. The cytosol was prepared from each strain and analyzed for the expression of Nap1p and Kap114-PrA by Western blotting (input). The same cytosol was used for immunoisolation of Kap114-PrA and associated proteins (IP). After being washed, the associated proteins were eluted with a buffer containing the indicated concentrations of MgCl 2 and then analyzed by Western blotting for Nap1p and Kap114-PrA. (d) Binding between MBP-Kap114p and the indicated GST proteins was analyzed as described for panel a in the presence or absence of core histones (500 nM) and RanQ69L (20 μM), as indicated. (e and f) The localization of the indicated GFP-tagged proteins was analyzed in wild-type yeast. Coincident Hoechst staining is also shown.

Techniques Used: Binding Assay, Concentration Assay, Mutagenesis, SDS Page, Staining, Western Blot, Plasmid Preparation, Expressing, Construct, Transformation Assay

Nap1p promotes the association of Kap114p with histones in the nucleus. H2A-PrA and associated proteins were isolated from nuclear extracts from the indicated strains with IgG-Sepharose. Fractions were eluted with a MgCl 2 step gradient and analyzed by Coomassie staining (CBB) or Western blotted (WB) and probed with an anti-Myc antibody.
Figure Legend Snippet: Nap1p promotes the association of Kap114p with histones in the nucleus. H2A-PrA and associated proteins were isolated from nuclear extracts from the indicated strains with IgG-Sepharose. Fractions were eluted with a MgCl 2 step gradient and analyzed by Coomassie staining (CBB) or Western blotted (WB) and probed with an anti-Myc antibody.

Techniques Used: Isolation, Staining, Western Blot

Kap114p associates with Nap1p and histones in the nucleus. (a) Kap114-PrA nuclear extract was incubated with IgG-Sepharose and then washed, and proteins were eluted with a MgCl 2 step gradient and then analyzed by Western blotting (WB) with an anti-Nap1p antibody. Molecular standards, in kilodaltons, are indicated on the left. (b) Equivalent amounts of cytosol and nuclear extracts from Kap114-PrA cells were analyzed by Western blotting with antibodies against Pgk1p or acetylated H4. H2A-PrA (c) and H4-PrA (d) and associated proteins were isolated from nuclear extracts and analyzed by Coomassie staining (CBB) or Western blotted with the indicated antibodies. The fractions shown in panels c and d were analyzed on the same gel and blot and were treated identically. Wash, final wash fraction; U/B, unbound fraction. (e) H2A-PrA and associated proteins were isolated from nuclear extracts as described for panel c, washed, incubated with 20 μM RanQ69L at room temperature for 1 h, washed again, eluted, and analyzed as described above. Wash-1, final wash fraction prior to RanQ69L addition; Wash-2, wash fraction prior to MgCl 2 elution. (f) Binding of MBP-Kap114p (250 nM) to GST-H2A 1-46 (1 μM) was assessed in the presence or absence of Nap1p (1 μM) and/or RanQ69L (20 μM).
Figure Legend Snippet: Kap114p associates with Nap1p and histones in the nucleus. (a) Kap114-PrA nuclear extract was incubated with IgG-Sepharose and then washed, and proteins were eluted with a MgCl 2 step gradient and then analyzed by Western blotting (WB) with an anti-Nap1p antibody. Molecular standards, in kilodaltons, are indicated on the left. (b) Equivalent amounts of cytosol and nuclear extracts from Kap114-PrA cells were analyzed by Western blotting with antibodies against Pgk1p or acetylated H4. H2A-PrA (c) and H4-PrA (d) and associated proteins were isolated from nuclear extracts and analyzed by Coomassie staining (CBB) or Western blotted with the indicated antibodies. The fractions shown in panels c and d were analyzed on the same gel and blot and were treated identically. Wash, final wash fraction; U/B, unbound fraction. (e) H2A-PrA and associated proteins were isolated from nuclear extracts as described for panel c, washed, incubated with 20 μM RanQ69L at room temperature for 1 h, washed again, eluted, and analyzed as described above. Wash-1, final wash fraction prior to RanQ69L addition; Wash-2, wash fraction prior to MgCl 2 elution. (f) Binding of MBP-Kap114p (250 nM) to GST-H2A 1-46 (1 μM) was assessed in the presence or absence of Nap1p (1 μM) and/or RanQ69L (20 μM).

Techniques Used: Incubation, Western Blot, Isolation, Staining, Binding Assay

Kap114p inhibits Nap1p-mediated chromatin assembly on plasmid templates. (a) Plasmid supercoiling assays were performed according to the schematic by the use of recombinant Nap1p (500 nM) in the presence of MBP-Kap114p or MBP- lacZ α (625 nM, 1.25 μM, or 2.5 μM), as indicated. After deproteinization, the DNAs were analyzed in an agarose gel. (b) Reactions were performed with Nap1p according to the schematic, incubated with MBP-Kap114p or MBP- lacZ α (625 nM, 1.25 μM, or 2.5 μM) for 1 h, and analyzed as described above. S, supercoiled DNA.
Figure Legend Snippet: Kap114p inhibits Nap1p-mediated chromatin assembly on plasmid templates. (a) Plasmid supercoiling assays were performed according to the schematic by the use of recombinant Nap1p (500 nM) in the presence of MBP-Kap114p or MBP- lacZ α (625 nM, 1.25 μM, or 2.5 μM), as indicated. After deproteinization, the DNAs were analyzed in an agarose gel. (b) Reactions were performed with Nap1p according to the schematic, incubated with MBP-Kap114p or MBP- lacZ α (625 nM, 1.25 μM, or 2.5 μM) for 1 h, and analyzed as described above. S, supercoiled DNA.

Techniques Used: Plasmid Preparation, Recombinant, Agarose Gel Electrophoresis, Incubation

17) Product Images from "Purification of phage display-modified bacteriophage T4 by affinity chromatography"

Article Title: Purification of phage display-modified bacteriophage T4 by affinity chromatography

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-11-59

Elution profile for the bacteriophage HAP1 modified with His tag and purified on Ni-NTA agarose compared to a non-modified phage . wash 1 - phage concentration in the washing flow-through (1 st litre of washing buffer) wash 2 - phage concentration in the washing flow-through (2 nd litre of washing buffer) wash 3 - phage concentration in the final washing fraction (3 rd litre of washing buffer) elution 1 - phage concentration in the first elution fraction (imidazole 100 mM) elution 2 - phage concentration in the second elution fraction (imidazole 200 mM) elution 3 - phage concentration in the third elution fraction (imidazole 300 mM) elution 4 - phage concentration in the fourth elution fraction (imidazole 400 mM) elution 5 - phage concentration in the fifth elution fraction (imidazole 500 mM)
Figure Legend Snippet: Elution profile for the bacteriophage HAP1 modified with His tag and purified on Ni-NTA agarose compared to a non-modified phage . wash 1 - phage concentration in the washing flow-through (1 st litre of washing buffer) wash 2 - phage concentration in the washing flow-through (2 nd litre of washing buffer) wash 3 - phage concentration in the final washing fraction (3 rd litre of washing buffer) elution 1 - phage concentration in the first elution fraction (imidazole 100 mM) elution 2 - phage concentration in the second elution fraction (imidazole 200 mM) elution 3 - phage concentration in the third elution fraction (imidazole 300 mM) elution 4 - phage concentration in the fourth elution fraction (imidazole 400 mM) elution 5 - phage concentration in the fifth elution fraction (imidazole 500 mM)

Techniques Used: Modification, Purification, Concentration Assay, Flow Cytometry

Elution profile for the bacteriophage HAP1 modified with GST tag and purified on glutathione Sepharose compared to a non-modified phage . wash 1 - phage concentration in the washing flow-through (1 st litre of washing buffer) wash 2 - phage concentration in the washing flow-through (2 nd litre of washing buffer) wash 3 - phage concentration in the final washing fraction (3 rd litre of washing buffer) elution 1 - phage concentration in the first elution fraction elution 2 - phage concentration in the second elution fraction elution 3 - phage concentration in the third elution fraction elution 4 - phage concentration in the fourth elution fraction (modified phage only) elution 5 - phage concentration in the fifth elution fraction (modified phage only)
Figure Legend Snippet: Elution profile for the bacteriophage HAP1 modified with GST tag and purified on glutathione Sepharose compared to a non-modified phage . wash 1 - phage concentration in the washing flow-through (1 st litre of washing buffer) wash 2 - phage concentration in the washing flow-through (2 nd litre of washing buffer) wash 3 - phage concentration in the final washing fraction (3 rd litre of washing buffer) elution 1 - phage concentration in the first elution fraction elution 2 - phage concentration in the second elution fraction elution 3 - phage concentration in the third elution fraction elution 4 - phage concentration in the fourth elution fraction (modified phage only) elution 5 - phage concentration in the fifth elution fraction (modified phage only)

Techniques Used: Modification, Purification, Concentration Assay, Flow Cytometry

Elution profile for the bacteriophage HAP1 modified with GST tag and purified on glutathione Sepharose compared to the same phage (HAP1) modified with a non-specific tag . wash - phage concentration in the final washing flow-through (4 th litre of washing buffer) elution 1 - phage concentration in the first elution fraction elution 2 - phage concentration in the second elution fraction elution 3 - phage concentration in the third elution fraction elution 4 - phage concentration in the fourth elution fraction
Figure Legend Snippet: Elution profile for the bacteriophage HAP1 modified with GST tag and purified on glutathione Sepharose compared to the same phage (HAP1) modified with a non-specific tag . wash - phage concentration in the final washing flow-through (4 th litre of washing buffer) elution 1 - phage concentration in the first elution fraction elution 2 - phage concentration in the second elution fraction elution 3 - phage concentration in the third elution fraction elution 4 - phage concentration in the fourth elution fraction

Techniques Used: Modification, Purification, Concentration Assay, Flow Cytometry

Elution profile for the bacteriophage HAP1 modified with GST tag and purified on Ni-NTA agarose compared to the same phage (HAP1) modified with a non-specific tag . wash 1 - phage concentration in the washing flow-through (1 st litre of washing buffer) wash 2 - phage concentration in the washing flow-through (2 nd litre of washing buffer) wash 3 - phage concentration in the washing flow-through (3 rd litre of washing buffer) wash 4 - phage concentration in the final washing fraction (4 th litre of washing buffer) elution 1 - phage concentration in the first elution fraction (imidazole 100 mM) elution 2 - phage concentration in the second elution fraction (imidazole 200 mM) elution 3 - phage concentration in the third elution fraction (imidazole 300 mM) elution 4 - phage concentration in the fourth elution fraction (imidazole 400 mM) elution 5 - phage concentration in the fifth elution fraction (imidazole 500 mM)
Figure Legend Snippet: Elution profile for the bacteriophage HAP1 modified with GST tag and purified on Ni-NTA agarose compared to the same phage (HAP1) modified with a non-specific tag . wash 1 - phage concentration in the washing flow-through (1 st litre of washing buffer) wash 2 - phage concentration in the washing flow-through (2 nd litre of washing buffer) wash 3 - phage concentration in the washing flow-through (3 rd litre of washing buffer) wash 4 - phage concentration in the final washing fraction (4 th litre of washing buffer) elution 1 - phage concentration in the first elution fraction (imidazole 100 mM) elution 2 - phage concentration in the second elution fraction (imidazole 200 mM) elution 3 - phage concentration in the third elution fraction (imidazole 300 mM) elution 4 - phage concentration in the fourth elution fraction (imidazole 400 mM) elution 5 - phage concentration in the fifth elution fraction (imidazole 500 mM)

Techniques Used: Modification, Purification, Concentration Assay, Flow Cytometry

18) Product Images from "Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation"

Article Title: Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

Journal: eLife

doi: 10.7554/eLife.21755

Fap7:uS11 facilitates RanGTP-dependent release of eS26 from Pse1. ( A ) eS26 and Fap7:uS11 interact with Pse1. Recombinant GST-Pse1 was immobilized on Glutathione Sepharose beads and incubated with buffer or eS26 alone. After washing away unbound proteins, beads were further incubated in presence of buffer or Fap7:uS11 for 1 hr and washed again. Beads were then eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 10% input. GST-baits are indicated with asterisks. ( B ) Fap7:uS11 facilitates RanGTP-dependent release of eS26. Immobilized GST-Pse1 was incubated first with eS26 alone (left panel). After washing away unbound proteins, beads were further incubated with RanGTP alone or RanGTP together with either Fap7:uS11 or Fap7:uS11 3R for indicated time points and washed again. Beads were then eluted and analyzed as described above. Right panel shows a schematic for how Fap7:uS11 facilitates RanGTP-dependent release of eS26 from Pse1. DOI: http://dx.doi.org/10.7554/eLife.21755.011
Figure Legend Snippet: Fap7:uS11 facilitates RanGTP-dependent release of eS26 from Pse1. ( A ) eS26 and Fap7:uS11 interact with Pse1. Recombinant GST-Pse1 was immobilized on Glutathione Sepharose beads and incubated with buffer or eS26 alone. After washing away unbound proteins, beads were further incubated in presence of buffer or Fap7:uS11 for 1 hr and washed again. Beads were then eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 10% input. GST-baits are indicated with asterisks. ( B ) Fap7:uS11 facilitates RanGTP-dependent release of eS26. Immobilized GST-Pse1 was incubated first with eS26 alone (left panel). After washing away unbound proteins, beads were further incubated with RanGTP alone or RanGTP together with either Fap7:uS11 or Fap7:uS11 3R for indicated time points and washed again. Beads were then eluted and analyzed as described above. Right panel shows a schematic for how Fap7:uS11 facilitates RanGTP-dependent release of eS26 from Pse1. DOI: http://dx.doi.org/10.7554/eLife.21755.011

Techniques Used: Recombinant, Incubation, SDS Page, Staining, Western Blot

Nuclear import of uS11 and Fap7. ( A ) A C-terminally GFP-tagged uS11 cannot complement the lethality of a rps14a∆rps14b∆ double knockout strain. The rps14a∆rps14b∆ pURA-uS11B shuffle strain was transformed with a plasmid encoding C-terminally tagged uS11-GFP and spotted in 10-fold dilutions on selective Leucine-deficient media plates with or without FOA to select against pURA-uS11B and grown at 30°C for 3–4 days. ( B ) Nuclear uptake of uS11-GFP is not impaired in kap123∆, msn5∆, kap114∆ sxm1∆ and kap120∆ sxm1∆ nmd5∆ mutants. Strains expressing uS11-GFP were grown in synthetic media at 25°C to mid-log phase. Ts -mutant strains were then shifted to 37°C for 4 hr and localization of uS11-GFP was analyzed by fluorescence microscopy. Scale bar = 5 µm. ( C ) Fap7:uS11, but not Fap7, interacts with Kap104. uS11, but not Fap7 interacts with Kap95, Kap114, Pdr6. Fap7 and uS11 alone, but not the Fap7:uS11 dimer interact with Kap123. Only Fap7 interacts with Msn5. Recombinant GST-tagged importins were immobilized on Glutathione Sepharose beads and incubated with buffer, purified Fap7 or Fap7:uS11 for 1 hr. After washing away unbound proteins, beads were eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 10% input. GST-baits are indicated with asterisks. ( D ) Nuclear uptake of GFP-Fap7 is not impaired in several importin mutants and upon depletion of uS11. Strains expressing GFP-Fap7 were grown in synthetic media at 25°C to mid-log phase. Ts -mutant strains were then shifted to 37°C for 4 hr while P Gal1 -RPS14A rps14b∆ was grown overnight at 25°C in repressive glucose medium. Localization of GFP-Fap7 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.21755.010
Figure Legend Snippet: Nuclear import of uS11 and Fap7. ( A ) A C-terminally GFP-tagged uS11 cannot complement the lethality of a rps14a∆rps14b∆ double knockout strain. The rps14a∆rps14b∆ pURA-uS11B shuffle strain was transformed with a plasmid encoding C-terminally tagged uS11-GFP and spotted in 10-fold dilutions on selective Leucine-deficient media plates with or without FOA to select against pURA-uS11B and grown at 30°C for 3–4 days. ( B ) Nuclear uptake of uS11-GFP is not impaired in kap123∆, msn5∆, kap114∆ sxm1∆ and kap120∆ sxm1∆ nmd5∆ mutants. Strains expressing uS11-GFP were grown in synthetic media at 25°C to mid-log phase. Ts -mutant strains were then shifted to 37°C for 4 hr and localization of uS11-GFP was analyzed by fluorescence microscopy. Scale bar = 5 µm. ( C ) Fap7:uS11, but not Fap7, interacts with Kap104. uS11, but not Fap7 interacts with Kap95, Kap114, Pdr6. Fap7 and uS11 alone, but not the Fap7:uS11 dimer interact with Kap123. Only Fap7 interacts with Msn5. Recombinant GST-tagged importins were immobilized on Glutathione Sepharose beads and incubated with buffer, purified Fap7 or Fap7:uS11 for 1 hr. After washing away unbound proteins, beads were eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 10% input. GST-baits are indicated with asterisks. ( D ) Nuclear uptake of GFP-Fap7 is not impaired in several importin mutants and upon depletion of uS11. Strains expressing GFP-Fap7 were grown in synthetic media at 25°C to mid-log phase. Ts -mutant strains were then shifted to 37°C for 4 hr while P Gal1 -RPS14A rps14b∆ was grown overnight at 25°C in repressive glucose medium. Localization of GFP-Fap7 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.21755.010

Techniques Used: Double Knockout, Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Fluorescence, Microscopy, Recombinant, Incubation, Purification, SDS Page, Staining, Western Blot

Fap7 ATPase activity organizes and recruits the uS11:eS26 subcomplex to helix 23 of 18S rRNA. ( A ) Schematic for ATP-dependent loading of uS11:eS26 onto helix 23 of 18S rRNA by Fap7. Asterisk indicates a potential intermediate Fap7-ATP:uS11:eS26 complex. ( B ) Recombinant GST-uS11 was immobilized on Glutathione Sepharose beads before incubation with Fap7 or Fap7-2, eS26, ATP or non-hydrolysable ATP analog AMP-PNP and 18S helix 23 rRNA. ( C ) Recombinant GST-eS26 was immobilized on Glutathione Sepharose beads before incubation with 18S helix 23 rRNA. After washing away unbound proteins and RNA, beads were eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. For analysis of RNA, samples were Phenol-extracted and separated by denaturing PAGE followed by GelRed staining. RNA was quantified with respect to the input. L = 10% input. GST-baits and pulled-down proteins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.21755.008
Figure Legend Snippet: Fap7 ATPase activity organizes and recruits the uS11:eS26 subcomplex to helix 23 of 18S rRNA. ( A ) Schematic for ATP-dependent loading of uS11:eS26 onto helix 23 of 18S rRNA by Fap7. Asterisk indicates a potential intermediate Fap7-ATP:uS11:eS26 complex. ( B ) Recombinant GST-uS11 was immobilized on Glutathione Sepharose beads before incubation with Fap7 or Fap7-2, eS26, ATP or non-hydrolysable ATP analog AMP-PNP and 18S helix 23 rRNA. ( C ) Recombinant GST-eS26 was immobilized on Glutathione Sepharose beads before incubation with 18S helix 23 rRNA. After washing away unbound proteins and RNA, beads were eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. For analysis of RNA, samples were Phenol-extracted and separated by denaturing PAGE followed by GelRed staining. RNA was quantified with respect to the input. L = 10% input. GST-baits and pulled-down proteins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.21755.008

Techniques Used: Activity Assay, Recombinant, Incubation, SDS Page, Staining, Western Blot, Polyacrylamide Gel Electrophoresis

19) Product Images from "A Ypt32p Exchange Factor Is a Putative Effector of Ypt1p"

Article Title: A Ypt32p Exchange Factor Is a Putative Effector of Ypt1p

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.01-12-0577

The Ypt32p exchange factor is a putative effector of Ypt1p. (A) Lysate, prepared from a wild-type yeast strain, was incubated with beads that contain GST-Ypt1p, GST-Ypt51p, or GST. The beads were either preloaded with GTPγS or GDP. The treated beads were incubated with [ 3 H]GDP-Ypt32p at 30°C for various periods of time. [ 3 H]GDP that bound to protein was measured by by filter-binding, and the data are expressed as the percentage of label bound to Ypt32p. (B) Beads containing 0.8 mg of GST-Ypt1p-GTPγS, GST-Ypt1p-GDP, or GST were incubated with a yeast lysate (800 mg) and then assayed for 30 min at room temperature in the presence of 1.6 nmol of (His 6 )-Ypt32p and 32 nmol of [ 35 S]GTPγS. The reaction was stopped by the addition of 1 ml of ice-cold stop buffer. The beads were spun, and the supernatant was incubated with 25 μl of packed Ni-NTA agarose beads for 1 h at 4°C. The Ni-NTA beads were washed three times with 1 ml of stop buffer, and the amount of GTPγS that bound to the beads was measured by filter-binding. The intrinsic uptake of GTPγS onto Ypt32p was measured in the presence of immobilized GST, and the value obtained was subtracted as background. (C) TRAPP-depleted cytosol was incubated with GST-Ypt1p-GTPγS immobilized on beads. The beads were then incubated with [ 3 H]GDP-Ypt32p or [ 3 H]GDP-Ypt1p for 20 min at 30°C. The intrinsic rate of [ 3 H]GDP release from Ypt32p or Ypt1p was measured in the presence of immobilized GST, and the value obtained was subtracted as background.
Figure Legend Snippet: The Ypt32p exchange factor is a putative effector of Ypt1p. (A) Lysate, prepared from a wild-type yeast strain, was incubated with beads that contain GST-Ypt1p, GST-Ypt51p, or GST. The beads were either preloaded with GTPγS or GDP. The treated beads were incubated with [ 3 H]GDP-Ypt32p at 30°C for various periods of time. [ 3 H]GDP that bound to protein was measured by by filter-binding, and the data are expressed as the percentage of label bound to Ypt32p. (B) Beads containing 0.8 mg of GST-Ypt1p-GTPγS, GST-Ypt1p-GDP, or GST were incubated with a yeast lysate (800 mg) and then assayed for 30 min at room temperature in the presence of 1.6 nmol of (His 6 )-Ypt32p and 32 nmol of [ 35 S]GTPγS. The reaction was stopped by the addition of 1 ml of ice-cold stop buffer. The beads were spun, and the supernatant was incubated with 25 μl of packed Ni-NTA agarose beads for 1 h at 4°C. The Ni-NTA beads were washed three times with 1 ml of stop buffer, and the amount of GTPγS that bound to the beads was measured by filter-binding. The intrinsic uptake of GTPγS onto Ypt32p was measured in the presence of immobilized GST, and the value obtained was subtracted as background. (C) TRAPP-depleted cytosol was incubated with GST-Ypt1p-GTPγS immobilized on beads. The beads were then incubated with [ 3 H]GDP-Ypt32p or [ 3 H]GDP-Ypt1p for 20 min at 30°C. The intrinsic rate of [ 3 H]GDP release from Ypt32p or Ypt1p was measured in the presence of immobilized GST, and the value obtained was subtracted as background.

Techniques Used: Incubation, Binding Assay

Depletion of TRAPP abolishes the Ypt1p exchange activity from cytosol. TRAPP was depleted by incubating IgG-Sepharose beads with cytosol prepared from a strain in which Bet3p is Protein A tagged and the DSS4 gene is deleted. Samples containing 0.2 mg of mock-treated or 0.5 mg of IgG-Sepharose–treated cytosol were resolved by SDS-PAGE and analyzed by Western blot analysis with α-Trs33p serum (C). TRAPP-depleted or mock-treated cytosol was incubated with 5 pmol of Ypt1p (A) or Ypt32p (B) preloaded with [ 3 H]GDP. At the time intervals indicated, radioactivity that bound to protein was determined by filter-binding, and the data are expressed as the percentage of label bound to Ypt1p or Ypt32p. The intrinsic rate of [ 3 H]GDP release from Ypt1p or Ypt32p was measured in the presence of BSA.
Figure Legend Snippet: Depletion of TRAPP abolishes the Ypt1p exchange activity from cytosol. TRAPP was depleted by incubating IgG-Sepharose beads with cytosol prepared from a strain in which Bet3p is Protein A tagged and the DSS4 gene is deleted. Samples containing 0.2 mg of mock-treated or 0.5 mg of IgG-Sepharose–treated cytosol were resolved by SDS-PAGE and analyzed by Western blot analysis with α-Trs33p serum (C). TRAPP-depleted or mock-treated cytosol was incubated with 5 pmol of Ypt1p (A) or Ypt32p (B) preloaded with [ 3 H]GDP. At the time intervals indicated, radioactivity that bound to protein was determined by filter-binding, and the data are expressed as the percentage of label bound to Ypt1p or Ypt32p. The intrinsic rate of [ 3 H]GDP release from Ypt1p or Ypt32p was measured in the presence of BSA.

Techniques Used: Activity Assay, SDS Page, Western Blot, Incubation, Radioactivity, Binding Assay

Depletion of TRAPP does not affect the Ypt32p exchange activity. (A) The P100 fraction of SFNY1088 was treated with buffer (control) or 0.5 M NaCl, centrifuged, and assayed as described below. To deplete TRAPP, the salt-treated supernatant (2.4 mg) was incubated with 20 μl of packed IgG-Sepharose or mock-treated with Sepharose 4B as described in MATERIALS AND METHODS. The TRAPP-depleted or mock-treated sample was incubated with [ 35 S]GTPγS for 30 min at room temperature in the presence or absence of (His 6 )-Ypt32p. Samples were then incubated with 20 μl of packed Nickel-nitrilotriacetic acid-agarose (Ni-NTA) beads for 1 h at 4°C, and GTPγS uptake was measured. The intrinsic rate of GTPγS uptake onto Ypt32p measured in the presence of BSA, and the value obtained in the absence of (His 6 )-Ypt32p was subtracted as background. (B) A P100 fraction, prepared from SFNY1088, was treated with buffer (control) or 0.5 M NaCl and centrifuged. The supernatant was incubated with IgG-Sepharose or mock treated with Sepharose 4B. The supernatants (s) and pellets (p) as well as the TRAPP-depleted and mock-treated samples were assayed at 30°C for 30 min for their ability to stimulate the release of [ 3 H]GDP from Ypt1p. The intrinsic rate of [ 3 H]GDP release from Ypt1p was measured in the presence of BSA, and the value obtained was subtracted as background. (C) TRAPP-depleted (0.5 mg) and mock-treated (0.3 mg) samples were resolved by SDS-PAGE and analyzed by Western blot analysis using antibodies directed against Trs120p, Bet3p, Trs33p, Trs31p, Trs20p, and Bet5p.
Figure Legend Snippet: Depletion of TRAPP does not affect the Ypt32p exchange activity. (A) The P100 fraction of SFNY1088 was treated with buffer (control) or 0.5 M NaCl, centrifuged, and assayed as described below. To deplete TRAPP, the salt-treated supernatant (2.4 mg) was incubated with 20 μl of packed IgG-Sepharose or mock-treated with Sepharose 4B as described in MATERIALS AND METHODS. The TRAPP-depleted or mock-treated sample was incubated with [ 35 S]GTPγS for 30 min at room temperature in the presence or absence of (His 6 )-Ypt32p. Samples were then incubated with 20 μl of packed Nickel-nitrilotriacetic acid-agarose (Ni-NTA) beads for 1 h at 4°C, and GTPγS uptake was measured. The intrinsic rate of GTPγS uptake onto Ypt32p measured in the presence of BSA, and the value obtained in the absence of (His 6 )-Ypt32p was subtracted as background. (B) A P100 fraction, prepared from SFNY1088, was treated with buffer (control) or 0.5 M NaCl and centrifuged. The supernatant was incubated with IgG-Sepharose or mock treated with Sepharose 4B. The supernatants (s) and pellets (p) as well as the TRAPP-depleted and mock-treated samples were assayed at 30°C for 30 min for their ability to stimulate the release of [ 3 H]GDP from Ypt1p. The intrinsic rate of [ 3 H]GDP release from Ypt1p was measured in the presence of BSA, and the value obtained was subtracted as background. (C) TRAPP-depleted (0.5 mg) and mock-treated (0.3 mg) samples were resolved by SDS-PAGE and analyzed by Western blot analysis using antibodies directed against Trs120p, Bet3p, Trs33p, Trs31p, Trs20p, and Bet5p.

Techniques Used: Activity Assay, Incubation, SDS Page, Western Blot

Chemically pure TRAPP stimulates GTPγS uptake onto Ypt1p but not Ypt32p. TRAPP was purified by affinity purification from a strain in which Trs33p was TAP tagged. Calmodulin agarose beads with or without TRAPP were incubated with 5 pmol of Ypt1p or Ypt32p at room temperature in the presence of [ 35 S]GTPγS. At the time intervals indicated, radioactivity that bound to protein was measured by the filter-binding assay described in MATERIALS AND METHODS. The data are expressed as picomoles of GTPγS retained on the filter.
Figure Legend Snippet: Chemically pure TRAPP stimulates GTPγS uptake onto Ypt1p but not Ypt32p. TRAPP was purified by affinity purification from a strain in which Trs33p was TAP tagged. Calmodulin agarose beads with or without TRAPP were incubated with 5 pmol of Ypt1p or Ypt32p at room temperature in the presence of [ 35 S]GTPγS. At the time intervals indicated, radioactivity that bound to protein was measured by the filter-binding assay described in MATERIALS AND METHODS. The data are expressed as picomoles of GTPγS retained on the filter.

Techniques Used: Purification, Affinity Purification, Incubation, Radioactivity, Filter-binding Assay

20) Product Images from "Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA"

Article Title: Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600050

Human and S. pombe Prp5 associate with U1 and U2 snRNPs. ( A ) SpPrp5-TAP association with pre-mRNA depends on both U1 and U2 snRNPs. Extracts prepared from strains containing SpPrp5-TAP, U1-A-TAP, or U2-A′-TAP were treated with RNase H alone (lane 2) or with RNase H and oligonucleotides targeting nt 1–14 of U1 (lane 3), nt 28–42 of U2 (lane 4), or nt 21–69 U6 snRNA as a control (lane 5). Extracts were then incubated with 32 P-labeled pre-mRNA +ATP, and complexes were affinity selected using IgG-sepharose to bind the TAP moiety. Copurifying pre-mRNA was analyzed by 10% PAGE (upper). Northern analysis of input snRNAs from SpPrp5-TAP extract after RNase H degradation (lower). The U1-A-TAP and U2-A′-TAP extracts were analyzed in parallel and showed similar levels of snRNA degradation (data not shown). ( B ) Affinity selection of snRNA associated with SpPrp5. Untagged (WT) or SpPrp5-TAP extracts were incubated at 30°C for 30 min and then with IgG-sepharose beads. Lanes 1 and 4, 1/4 of input. Copurifying snRNAs were analyzed by Northern blotting. ( C ) Affinity selection of U1 and U2 snRNP proteins by SpPrp5. Extracts from doubly tagged S. pombe strains containing SpPrp5-TAP and either U1-70K-3HA or U2-A′-3HA were incubated with IgG-sepharose. Copurifying proteins were analyzed by Western blotting using anti-HA antibodies. Lanes 1–2 and 7–8: 1/4 of the input extracts. Lanes 3–4 and 9–10: IgG-selected material from cells lacking any TAP-tagged protein as a control. Lanes 5–6 and 11–12: HA-tagged proteins copurifying with SpPrp5-TAP. ( D ) hPrp5 co-IPs U1 and U2 snRNPs. HeLa nuclear extract was incubated with –/+ATP as indicated, and then with protein A-bound antibodies. 1/20 of input (lane 1), beads alone (lanes 2–3), anti-hPrp5 #1 against a C-terminal peptide (lanes 4–5), anti-hPrp5 #2 against the internal DPLD motif (lanes 6–7).
Figure Legend Snippet: Human and S. pombe Prp5 associate with U1 and U2 snRNPs. ( A ) SpPrp5-TAP association with pre-mRNA depends on both U1 and U2 snRNPs. Extracts prepared from strains containing SpPrp5-TAP, U1-A-TAP, or U2-A′-TAP were treated with RNase H alone (lane 2) or with RNase H and oligonucleotides targeting nt 1–14 of U1 (lane 3), nt 28–42 of U2 (lane 4), or nt 21–69 U6 snRNA as a control (lane 5). Extracts were then incubated with 32 P-labeled pre-mRNA +ATP, and complexes were affinity selected using IgG-sepharose to bind the TAP moiety. Copurifying pre-mRNA was analyzed by 10% PAGE (upper). Northern analysis of input snRNAs from SpPrp5-TAP extract after RNase H degradation (lower). The U1-A-TAP and U2-A′-TAP extracts were analyzed in parallel and showed similar levels of snRNA degradation (data not shown). ( B ) Affinity selection of snRNA associated with SpPrp5. Untagged (WT) or SpPrp5-TAP extracts were incubated at 30°C for 30 min and then with IgG-sepharose beads. Lanes 1 and 4, 1/4 of input. Copurifying snRNAs were analyzed by Northern blotting. ( C ) Affinity selection of U1 and U2 snRNP proteins by SpPrp5. Extracts from doubly tagged S. pombe strains containing SpPrp5-TAP and either U1-70K-3HA or U2-A′-3HA were incubated with IgG-sepharose. Copurifying proteins were analyzed by Western blotting using anti-HA antibodies. Lanes 1–2 and 7–8: 1/4 of the input extracts. Lanes 3–4 and 9–10: IgG-selected material from cells lacking any TAP-tagged protein as a control. Lanes 5–6 and 11–12: HA-tagged proteins copurifying with SpPrp5-TAP. ( D ) hPrp5 co-IPs U1 and U2 snRNPs. HeLa nuclear extract was incubated with –/+ATP as indicated, and then with protein A-bound antibodies. 1/20 of input (lane 1), beads alone (lanes 2–3), anti-hPrp5 #1 against a C-terminal peptide (lanes 4–5), anti-hPrp5 #2 against the internal DPLD motif (lanes 6–7).

Techniques Used: Incubation, Labeling, Polyacrylamide Gel Electrophoresis, Northern Blot, Selection, Western Blot

Different domains of SpPrp5 interact with U1 and U2 snRNP. ( A ) Schematic of GST-Prp5 deletion proteins. ( B ) ATPase activities of GST-SpPrp5 and deletion constructs, expressed and purified from E. coli . Protein A: full-length SpPrp5; B: aa171–1014; C: aa272–1014; D: aa427–1014; E: aa1–170; F: aa171–426. ( C ) Affinity selection of snRNA by GST-SpPrp5. Extracts were incubated with full-length or deleted SpPrp5 proteins as indicated, and then with glutathione-sepharose beads. Copurifying snRNAs were analyzed by Northern blotting. ( D ) Ability of deletion mutants to complement complex A formation in SpPrp5-TAP depleted extracts. Intact and depleted SpPrp5-TAP extracts were incubated with 32 P-labeled pre-mRNA as indicated. Either GST-FL-SpPrp5 (A) or deletion proteins (B–F) were added to depleted extracts.
Figure Legend Snippet: Different domains of SpPrp5 interact with U1 and U2 snRNP. ( A ) Schematic of GST-Prp5 deletion proteins. ( B ) ATPase activities of GST-SpPrp5 and deletion constructs, expressed and purified from E. coli . Protein A: full-length SpPrp5; B: aa171–1014; C: aa272–1014; D: aa427–1014; E: aa1–170; F: aa171–426. ( C ) Affinity selection of snRNA by GST-SpPrp5. Extracts were incubated with full-length or deleted SpPrp5 proteins as indicated, and then with glutathione-sepharose beads. Copurifying snRNAs were analyzed by Northern blotting. ( D ) Ability of deletion mutants to complement complex A formation in SpPrp5-TAP depleted extracts. Intact and depleted SpPrp5-TAP extracts were incubated with 32 P-labeled pre-mRNA as indicated. Either GST-FL-SpPrp5 (A) or deletion proteins (B–F) were added to depleted extracts.

Techniques Used: Construct, Purification, Selection, Incubation, Northern Blot, Labeling

SpPrp5 is required for the formation of S. pombe complex A. ( A ) Comparison of human (upper) and fission yeast (lower) Prp5 to S. cerevisiae ). Other regions of similarity are boxed in gray. * , DPLD motif unique to Prp5 homologs; RS/RD, region rich in RS, RD, and RE dipeptides. ( B ) (Upper) Schematic of SpPrp5-TAP fusion protein. CBP, calmodulin-binding peptide; TEV, TEV protease cleavage site; prot. A, two copies of the protein A IgG binding domain. (Lower) Analysis of SpPrp5 depletion. Untagged extracts (WT; lane 1), mock-depleted WT (lane 2), SpPrp5-TAP tagged (lane 3), and SpPrp5-TAP depleted (lane 4) were separated on 10% SDS–PAGE, Western blotted, and probed for TAP and for U2-SF3b155 as a control. Parallel Northern analysis indicated that no snRNAs were codepleted under these conditions (not shown). ( C ) GST-SpPrp5 reconstitutes SpPrp5-TAP depleted extracts. Intact and depleted SpPrp5-TAP extracts were incubated with 32 P-labeled pre-mRNA –/+ATP at 30°C as indicated. GST alone, GST-SpPrp5, or Prp43p were added to depleted extracts. Samples were separated on a native 4% polyacrylamide gel, and visualized by phosphorimaging. A, U2 snRNP complexes containing pre-mRNA; H, nonspecific complexes. ( D ) SpPrp5-TAP is present in complexes with pre-mRNA. Extracts prepared from strains containing SpPrp5-TAP, U2-A′-TAP, or no tagged protein were incubated with 32 P-labeled pre-mRNA –/+ATP as indicated, and complexes were affinity selected using IgG-sepharose to bind the TAP moiety. Copurifying pre-mRNA (upper); IgG-selected TAP-tagged proteins detected by Western analysis for the TAP tag (lower).
Figure Legend Snippet: SpPrp5 is required for the formation of S. pombe complex A. ( A ) Comparison of human (upper) and fission yeast (lower) Prp5 to S. cerevisiae ). Other regions of similarity are boxed in gray. * , DPLD motif unique to Prp5 homologs; RS/RD, region rich in RS, RD, and RE dipeptides. ( B ) (Upper) Schematic of SpPrp5-TAP fusion protein. CBP, calmodulin-binding peptide; TEV, TEV protease cleavage site; prot. A, two copies of the protein A IgG binding domain. (Lower) Analysis of SpPrp5 depletion. Untagged extracts (WT; lane 1), mock-depleted WT (lane 2), SpPrp5-TAP tagged (lane 3), and SpPrp5-TAP depleted (lane 4) were separated on 10% SDS–PAGE, Western blotted, and probed for TAP and for U2-SF3b155 as a control. Parallel Northern analysis indicated that no snRNAs were codepleted under these conditions (not shown). ( C ) GST-SpPrp5 reconstitutes SpPrp5-TAP depleted extracts. Intact and depleted SpPrp5-TAP extracts were incubated with 32 P-labeled pre-mRNA –/+ATP at 30°C as indicated. GST alone, GST-SpPrp5, or Prp43p were added to depleted extracts. Samples were separated on a native 4% polyacrylamide gel, and visualized by phosphorimaging. A, U2 snRNP complexes containing pre-mRNA; H, nonspecific complexes. ( D ) SpPrp5-TAP is present in complexes with pre-mRNA. Extracts prepared from strains containing SpPrp5-TAP, U2-A′-TAP, or no tagged protein were incubated with 32 P-labeled pre-mRNA –/+ATP as indicated, and complexes were affinity selected using IgG-sepharose to bind the TAP moiety. Copurifying pre-mRNA (upper); IgG-selected TAP-tagged proteins detected by Western analysis for the TAP tag (lower).

Techniques Used: Binding Assay, SDS Page, Western Blot, Northern Blot, Incubation, Labeling

21) Product Images from "Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus "

Article Title: Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.02117-07

Activation of p38 MAPK by anisomycin enhances translocation of FGF1. (A) In vivo phosphorylation of exogenously added FGF1 in NIH 3T3 cells was analyzed in the presence of increasing micromolar concentrations of anisomycin or 10 nM bafilomycin A1 (Baf.A1) as indicated. In vivo phosphorylated FGF1 ( 33 P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection. (B) NIH 3T3 cells were incubated with in vitro-labeled 35 S-FGF1 and heparin for 6 h in the presence of increasing concentrations of anisomycin as indicated. The cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (C) In vivo phosphorylation of FGF1 was analyzed in the absence or presence of 10 μM anisomycin, 1 μM Gö6976, 5 μM rottlerin, or 10 nM bafilomycin A1 as indicated. (D) NIH 3T3 cells were treated as explained for panel B but in the absence or presence of 10 μM anisomycin, 1 μM Gö6976, or 5 μM rottlerin as indicated. (E) The activity of PKCδ in the presence of various inhibitors was tested in vitro. Lysates of NIH 3T3 were subjected to immunoprecipitation (IP) using anti-Sumo-1 antibody (ctrl; lane 1) or anti-PKCδ antibody (lanes 2 to 7) in the absence (lane1 to 6) or presence (lane 7) of anti-PKCδ antibody-blocking peptide. The immunoprecipitated material was incubated in kinase buffer with MBP and [γ- 33 P]ATP, in the presence or absence of 10 μM SB203580, 10 μM anisomycin, 10 μM PD169316, 1 μM Gö6976, or 5 μM rottlerin, and thereafter the samples were analyzed by SDS-PAGE, fluorography ( 33 P-MBP), and Western blotting using anti-MBP antibody (total MBP).
Figure Legend Snippet: Activation of p38 MAPK by anisomycin enhances translocation of FGF1. (A) In vivo phosphorylation of exogenously added FGF1 in NIH 3T3 cells was analyzed in the presence of increasing micromolar concentrations of anisomycin or 10 nM bafilomycin A1 (Baf.A1) as indicated. In vivo phosphorylated FGF1 ( 33 P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection. (B) NIH 3T3 cells were incubated with in vitro-labeled 35 S-FGF1 and heparin for 6 h in the presence of increasing concentrations of anisomycin as indicated. The cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (C) In vivo phosphorylation of FGF1 was analyzed in the absence or presence of 10 μM anisomycin, 1 μM Gö6976, 5 μM rottlerin, or 10 nM bafilomycin A1 as indicated. (D) NIH 3T3 cells were treated as explained for panel B but in the absence or presence of 10 μM anisomycin, 1 μM Gö6976, or 5 μM rottlerin as indicated. (E) The activity of PKCδ in the presence of various inhibitors was tested in vitro. Lysates of NIH 3T3 were subjected to immunoprecipitation (IP) using anti-Sumo-1 antibody (ctrl; lane 1) or anti-PKCδ antibody (lanes 2 to 7) in the absence (lane1 to 6) or presence (lane 7) of anti-PKCδ antibody-blocking peptide. The immunoprecipitated material was incubated in kinase buffer with MBP and [γ- 33 P]ATP, in the presence or absence of 10 μM SB203580, 10 μM anisomycin, 10 μM PD169316, 1 μM Gö6976, or 5 μM rottlerin, and thereafter the samples were analyzed by SDS-PAGE, fluorography ( 33 P-MBP), and Western blotting using anti-MBP antibody (total MBP).

Techniques Used: Activation Assay, Translocation Assay, In Vivo, Immunodetection, Incubation, In Vitro, Labeling, Binding Assay, SDS Page, Activity Assay, Immunoprecipitation, Blocking Assay, Western Blot

Inhibition of p38 MAPK inhibits translocation of FGF1 to the cytosol and nucleus. (A) In vivo phosphorylation of exogenously added FGF1 in NIH 3T3 cells was analyzed in the absence or presence of micromolar concentrations of SB203580 or 10 nM bafilomycin A1 (Baf.A1), as indicated. In vivo-phosphorylated FGF1 ( 33 P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection. (B) NIH 3T3 cells were incubated with or without 5 μM anisomycin in the absence or presence of micromolar concentrations of SB203580 as indicated. Total cell lysates were analyzed by Western blotting using an anti-phospho-MK2-specific antibody (p-MK2) and an anti-total MK2 antibody. (C) The in vivo phosphorylation of FGF1 was analyzed in the presence of micromolar concentrations of PD169316 or SB202474 or 10 nM bafilomycin A1 as indicated. (D) In vivo phosphorylation of FGF1 was analyzed in the absence or presence of 5 μM SB203580, 20 nM TPA, or 10 nM bafilomycin A1 as indicated. (E) NIH 3T3 cells were incubated with in vitro-labeled 35 S-FGF1 and heparin for 6 h in the absence or presence of micromolar concentrations of SB203580 or 10 nM bafilomycin A1 as indicated. The cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (F) The subcellular fractions obtained by fractionation as for panel E were tested for the presence of the membrane-associated proteins Rab5a and calreticulin, the cytosolic protein ERK1/2, and the nuclear protein lamin A by specific antibodies.
Figure Legend Snippet: Inhibition of p38 MAPK inhibits translocation of FGF1 to the cytosol and nucleus. (A) In vivo phosphorylation of exogenously added FGF1 in NIH 3T3 cells was analyzed in the absence or presence of micromolar concentrations of SB203580 or 10 nM bafilomycin A1 (Baf.A1), as indicated. In vivo-phosphorylated FGF1 ( 33 P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection. (B) NIH 3T3 cells were incubated with or without 5 μM anisomycin in the absence or presence of micromolar concentrations of SB203580 as indicated. Total cell lysates were analyzed by Western blotting using an anti-phospho-MK2-specific antibody (p-MK2) and an anti-total MK2 antibody. (C) The in vivo phosphorylation of FGF1 was analyzed in the presence of micromolar concentrations of PD169316 or SB202474 or 10 nM bafilomycin A1 as indicated. (D) In vivo phosphorylation of FGF1 was analyzed in the absence or presence of 5 μM SB203580, 20 nM TPA, or 10 nM bafilomycin A1 as indicated. (E) NIH 3T3 cells were incubated with in vitro-labeled 35 S-FGF1 and heparin for 6 h in the absence or presence of micromolar concentrations of SB203580 or 10 nM bafilomycin A1 as indicated. The cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (F) The subcellular fractions obtained by fractionation as for panel E were tested for the presence of the membrane-associated proteins Rab5a and calreticulin, the cytosolic protein ERK1/2, and the nuclear protein lamin A by specific antibodies.

Techniques Used: Inhibition, Translocation Assay, In Vivo, Immunodetection, Incubation, Western Blot, In Vitro, Labeling, Binding Assay, SDS Page, Fractionation

siRNA knockdown of p38α inhibits translocation of FGF1. (A to C) NIH 3T3 cells were transfected with control siRNA (ctrl) or three different siRNAs specific for mouse p38α (α 1 , α 2 , and α 3 ). (A) Total cell lysates were analyzed for p38α (i) and total p38 MAPK (ii) by Western blotting. In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells. Phosphorylated FGF1 ( 33 P-FGF1) was detected by fluorography (iii), and the total cellular uptake was detected by anti-FGF1 immunodetection (total FGF1 [iv]). (B) In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells in the presence of 10 μM anisomycin. (C) In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells in the absence (i) or presence (ii to v) of LMB, and the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions before extraction of FGF1. (D) Nuclear and cytoplasmic fractions of NIH 3T3 cells obtained by the cellular fractionation method described for panel C were tested for the presence of the cytoplasmic proteins rab5a, calreticulin, and ERK1/2 and the nuclear protein lamin A by specific antibodies. (E and F) BJ cells were mock transfected or transfected with control siRNA (ctrl), two different siRNAs specific for human p38α (α1 and α2), or two different siRNAs specific for human p38β (β1 and β2). (E) The transfected cells were lysed and tested for the amount of p38α (i), p38β (ii), and ERK1/2 (loading control, [iii]) by immunoblotting. Translocation of FGF1 was tested in the transfected cells in an in vivo FGF1 phosphorylation assay. Phosphorylated FGF1 ( 33 P-FGF1) was detected by fluorography (iv), and the total cellular uptake was detected by anti-FGF1 immunodetection (total FGF1 [v]). No FGF1 was added in the first lane, panels iv and v. (F) siRNA-transfected BJ cells were incubated with in vitro-labeled 35 S-FGF1 and heparin for 6 h, and then the cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (G) The subcellular fractions obtained by fractionation as described for panel F were tested for the presence of the membrane-associated proteins Rab5a and calreticulin, the cytosolic protein ERK1/2, and the nuclear protein lamin A by specific antibodies.
Figure Legend Snippet: siRNA knockdown of p38α inhibits translocation of FGF1. (A to C) NIH 3T3 cells were transfected with control siRNA (ctrl) or three different siRNAs specific for mouse p38α (α 1 , α 2 , and α 3 ). (A) Total cell lysates were analyzed for p38α (i) and total p38 MAPK (ii) by Western blotting. In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells. Phosphorylated FGF1 ( 33 P-FGF1) was detected by fluorography (iii), and the total cellular uptake was detected by anti-FGF1 immunodetection (total FGF1 [iv]). (B) In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells in the presence of 10 μM anisomycin. (C) In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells in the absence (i) or presence (ii to v) of LMB, and the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions before extraction of FGF1. (D) Nuclear and cytoplasmic fractions of NIH 3T3 cells obtained by the cellular fractionation method described for panel C were tested for the presence of the cytoplasmic proteins rab5a, calreticulin, and ERK1/2 and the nuclear protein lamin A by specific antibodies. (E and F) BJ cells were mock transfected or transfected with control siRNA (ctrl), two different siRNAs specific for human p38α (α1 and α2), or two different siRNAs specific for human p38β (β1 and β2). (E) The transfected cells were lysed and tested for the amount of p38α (i), p38β (ii), and ERK1/2 (loading control, [iii]) by immunoblotting. Translocation of FGF1 was tested in the transfected cells in an in vivo FGF1 phosphorylation assay. Phosphorylated FGF1 ( 33 P-FGF1) was detected by fluorography (iv), and the total cellular uptake was detected by anti-FGF1 immunodetection (total FGF1 [v]). No FGF1 was added in the first lane, panels iv and v. (F) siRNA-transfected BJ cells were incubated with in vitro-labeled 35 S-FGF1 and heparin for 6 h, and then the cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (G) The subcellular fractions obtained by fractionation as described for panel F were tested for the presence of the membrane-associated proteins Rab5a and calreticulin, the cytosolic protein ERK1/2, and the nuclear protein lamin A by specific antibodies.

Techniques Used: Translocation Assay, Transfection, Western Blot, In Vivo, Immunodetection, Cell Fractionation, Phosphorylation Assay, Incubation, In Vitro, Labeling, Binding Assay, SDS Page, Fractionation

22) Product Images from "A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking"

Article Title: A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7601336

PACS-1 is required for CI-MPR function. ( A–C ) GST, GST-CI-MPR CD preincubated or not with CK2, or GST-CI-MPR CD containing the indicated mutations was incubated with Trx-PACS-1 FBR (residues 117–294) or Trx-GGA3 VHS+GAT , isolated with glutathione sepharose, washed three times with GST-binding buffer containing 4% NP-40 and analyzed by Western blot using anti-Trx (upper panels). Input of each GST-protein is shown (lower panel). GST-CI-MPR CD pulled down ∼1% of the Trx-PACS-1 FBR . ( D ) A7 cells were treated with scrambled (scr.) or PACS-1 siRNAs and cell lysates analyzed by Western blot using anti-PACS-1 or anti-tubulin. ( E ) A7 cells were treated with the indicated siRNA and Cathepsin D pulse chase experiments performed. Cellular and secreted fractions were immunoprecipitated with anti-cathepsin D and analyzed by fluorography. Precursor (P), intermediate (I) and mature (M) forms of cathepsin D are shown (lower panel). The percentage of missorted (secreted) cathepsin D compared to the processed form is shown ( n =3, P =0.01).
Figure Legend Snippet: PACS-1 is required for CI-MPR function. ( A–C ) GST, GST-CI-MPR CD preincubated or not with CK2, or GST-CI-MPR CD containing the indicated mutations was incubated with Trx-PACS-1 FBR (residues 117–294) or Trx-GGA3 VHS+GAT , isolated with glutathione sepharose, washed three times with GST-binding buffer containing 4% NP-40 and analyzed by Western blot using anti-Trx (upper panels). Input of each GST-protein is shown (lower panel). GST-CI-MPR CD pulled down ∼1% of the Trx-PACS-1 FBR . ( D ) A7 cells were treated with scrambled (scr.) or PACS-1 siRNAs and cell lysates analyzed by Western blot using anti-PACS-1 or anti-tubulin. ( E ) A7 cells were treated with the indicated siRNA and Cathepsin D pulse chase experiments performed. Cellular and secreted fractions were immunoprecipitated with anti-cathepsin D and analyzed by fluorography. Precursor (P), intermediate (I) and mature (M) forms of cathepsin D are shown (lower panel). The percentage of missorted (secreted) cathepsin D compared to the processed form is shown ( n =3, P =0.01).

Techniques Used: Incubation, Isolation, Binding Assay, Western Blot, Pulse Chase, Immunoprecipitation

PACS-1 binding to CK2β activates CK2. ( A ) Yeast transformed with the indicated Gal4 activation and DNA-binding domain (Gal4ad and Gal4bd) constructs were screened for growth on His + and His − media. ( B ) GST, GST-PACS-1 FBR (residues 117–266) or GST-PACS-1 FBR–CKmut was incubated with Trx-CK2β, isolated with glutathione sepharose, washed twice with GST-binding buffer, once with GST-binding buffer containing 1% deoxycholate and analyzed by Western blot using anti-Trx (upper panel). Input of each GST-protein is shown (lower panel). GST-PACS-1 FBR captured 2.5% of the input Trx-CK2β. ( C ) A7 cells infected with VV:WT or VV expressing HA-PACS-1 or HA-PACS-1 CKmut were lysed, immunoprecipitated with HA antibody and any co-immunoprecipitating CK2α and CK2β detected by Western blot using subunit-specific antisera (upper panels). HA-PACS-1 expression is shown (bottom panel). ( D ) In vitro CK2 holoenzyme activity assayed in the absence or presence of purified GST, GST-PACS-1 FBR (residues 117–266) or GST-PACS-1 FBR–CKmut . Activity is normalized to a parallel sample assayed in the absence of added protein. Error bars represent mean and s.d. of three independent experiments. ( E ) A7 cells were infected with VV:WT or VV expressing HA-PACS-1, HA-PACS-1 CKmut , HA-PACS-1 S278A or HA-PACS-1 S278A/CKmut and metabolically labeled with 32 P i . HA-proteins were immunoprecipitated with mAb HA.11, resolved by SDS–PAGE and analyzed by autoradiography (upper panel). HA-PACS-1 expression is shown (bottom panel). Error bars represent mean and s.d. of three independent experiments.
Figure Legend Snippet: PACS-1 binding to CK2β activates CK2. ( A ) Yeast transformed with the indicated Gal4 activation and DNA-binding domain (Gal4ad and Gal4bd) constructs were screened for growth on His + and His − media. ( B ) GST, GST-PACS-1 FBR (residues 117–266) or GST-PACS-1 FBR–CKmut was incubated with Trx-CK2β, isolated with glutathione sepharose, washed twice with GST-binding buffer, once with GST-binding buffer containing 1% deoxycholate and analyzed by Western blot using anti-Trx (upper panel). Input of each GST-protein is shown (lower panel). GST-PACS-1 FBR captured 2.5% of the input Trx-CK2β. ( C ) A7 cells infected with VV:WT or VV expressing HA-PACS-1 or HA-PACS-1 CKmut were lysed, immunoprecipitated with HA antibody and any co-immunoprecipitating CK2α and CK2β detected by Western blot using subunit-specific antisera (upper panels). HA-PACS-1 expression is shown (bottom panel). ( D ) In vitro CK2 holoenzyme activity assayed in the absence or presence of purified GST, GST-PACS-1 FBR (residues 117–266) or GST-PACS-1 FBR–CKmut . Activity is normalized to a parallel sample assayed in the absence of added protein. Error bars represent mean and s.d. of three independent experiments. ( E ) A7 cells were infected with VV:WT or VV expressing HA-PACS-1, HA-PACS-1 CKmut , HA-PACS-1 S278A or HA-PACS-1 S278A/CKmut and metabolically labeled with 32 P i . HA-proteins were immunoprecipitated with mAb HA.11, resolved by SDS–PAGE and analyzed by autoradiography (upper panel). HA-PACS-1 expression is shown (bottom panel). Error bars represent mean and s.d. of three independent experiments.

Techniques Used: Binding Assay, Transformation Assay, Activation Assay, Construct, Incubation, Isolation, Western Blot, Infection, Expressing, Immunoprecipitation, In Vitro, Activity Assay, Purification, Metabolic Labelling, Labeling, SDS Page, Autoradiography

PACS-1 binds to GGA3. ( A ), the middle region (MR), which contains the autoregulatory acidic cluster and Ser 278 ), and the C-terminal region (CTR) and PACS-1 cargo. ( B ) Diagram of GGA3 showing the VHS (Vps27, Hrs, Stam) domain, which binds to cargo proteins, the GAT (GGA and TOM) domain, which binds to ARF1, the hinge segment, which contains the autoregulatory acidic-dileucine motif and Ser 388 ) and GGA3 cargo. ( C ) Endogenous PACS-1 was immunoprecipitated from rat brain (lower panel) using anti-PACS-1 701 or 703, anti-PACS-2 834 or control IgG and co-precipitating GGA3 analyzed by SDS–PAGE and Western blot (upper panel). Immunoprecipitated PACS-1 and PACS-2 are shown by Western blot (bottom panel). ( D–G ) The indicated GST-fusion proteins were incubated with the indicated Trx-fusion proteins or with purified AP-1, isolated with glutathione sepharose and analyzed by Western blot using anti-Trx or anti-γ-adaptin antibody (upper panels). Each GST-protein is also shown (lower panel). The Trx-PACS-1 FBR (residues 117–294) band is shifted lower in (E) because Trx-PACS-1 FBR migrates at the same size as GST-GGA3 VHS . GST-GGA3 VHS−GAT captured ∼1% of Trx-PACS-1 FBR input, and GST-PACS-1 FBR (residues 117–294) captured ∼1%, 0.5% and 1% of the Trx-GGA3 VHS−GAT , γ-adaptin and Trx-CI-MPR cd input, respectively. Binding assays were conducted as described in Materials and methods, except 4% NP40 was used. ( H ) A7 cells infected with wild-type (WT) AV or AV expressing Myc-GGA3, Myc-GGA3 and HA-PACS-1, or Myc-GGA3 and HA-PACS-1 GGAmut were harvested and HA-tagged proteins immunoprecipitated and co-precipitating myc-GGA analyzed by Western blot (upper panel). Lower panels show myc-GGA3 and HA-PACS-1 expression.
Figure Legend Snippet: PACS-1 binds to GGA3. ( A ), the middle region (MR), which contains the autoregulatory acidic cluster and Ser 278 ), and the C-terminal region (CTR) and PACS-1 cargo. ( B ) Diagram of GGA3 showing the VHS (Vps27, Hrs, Stam) domain, which binds to cargo proteins, the GAT (GGA and TOM) domain, which binds to ARF1, the hinge segment, which contains the autoregulatory acidic-dileucine motif and Ser 388 ) and GGA3 cargo. ( C ) Endogenous PACS-1 was immunoprecipitated from rat brain (lower panel) using anti-PACS-1 701 or 703, anti-PACS-2 834 or control IgG and co-precipitating GGA3 analyzed by SDS–PAGE and Western blot (upper panel). Immunoprecipitated PACS-1 and PACS-2 are shown by Western blot (bottom panel). ( D–G ) The indicated GST-fusion proteins were incubated with the indicated Trx-fusion proteins or with purified AP-1, isolated with glutathione sepharose and analyzed by Western blot using anti-Trx or anti-γ-adaptin antibody (upper panels). Each GST-protein is also shown (lower panel). The Trx-PACS-1 FBR (residues 117–294) band is shifted lower in (E) because Trx-PACS-1 FBR migrates at the same size as GST-GGA3 VHS . GST-GGA3 VHS−GAT captured ∼1% of Trx-PACS-1 FBR input, and GST-PACS-1 FBR (residues 117–294) captured ∼1%, 0.5% and 1% of the Trx-GGA3 VHS−GAT , γ-adaptin and Trx-CI-MPR cd input, respectively. Binding assays were conducted as described in Materials and methods, except 4% NP40 was used. ( H ) A7 cells infected with wild-type (WT) AV or AV expressing Myc-GGA3, Myc-GGA3 and HA-PACS-1, or Myc-GGA3 and HA-PACS-1 GGAmut were harvested and HA-tagged proteins immunoprecipitated and co-precipitating myc-GGA analyzed by Western blot (upper panel). Lower panels show myc-GGA3 and HA-PACS-1 expression.

Techniques Used: Immunoprecipitation, SDS Page, Western Blot, Incubation, Purification, Isolation, Binding Assay, Infection, Expressing

23) Product Images from "Ric1-Rgp1 Complex Is a Guanine Nucleotide Exchange Factor for the Late Golgi Rab6A GTPase and an Effector of the Medial Golgi Rab33B GTPase *"

Article Title: Ric1-Rgp1 Complex Is a Guanine Nucleotide Exchange Factor for the Late Golgi Rab6A GTPase and an Effector of the Medial Golgi Rab33B GTPase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.414565

Rgp1 and Ric1 interact in vivo . A , HEK293T cells were transfected with either GFP or GFP-Rgp1 with Myc-Ric1 for 24 h, and Rgp1 was immunoprecipitated ( IP ) with anti-GFP-binding protein-conjugated agarose followed by immunoblotting with anti-Myc antibody
Figure Legend Snippet: Rgp1 and Ric1 interact in vivo . A , HEK293T cells were transfected with either GFP or GFP-Rgp1 with Myc-Ric1 for 24 h, and Rgp1 was immunoprecipitated ( IP ) with anti-GFP-binding protein-conjugated agarose followed by immunoblotting with anti-Myc antibody

Techniques Used: In Vivo, Transfection, Immunoprecipitation, Binding Assay

Rgp1 and Ric1-720–1344 interact directly with Rab6A-GDP in vitro . A , GST, GST-Rgp1, or GST-Ric1-720–1344 proteins were immobilized on glutathione-Sepharose followed by incubation with His-Rab6A T27N protein. Bound material was analyzed
Figure Legend Snippet: Rgp1 and Ric1-720–1344 interact directly with Rab6A-GDP in vitro . A , GST, GST-Rgp1, or GST-Ric1-720–1344 proteins were immobilized on glutathione-Sepharose followed by incubation with His-Rab6A T27N protein. Bound material was analyzed

Techniques Used: In Vitro, Incubation

Ric1-Rgp1 complex has guanine nucleotide exchange activity toward Rab6A in vitro . A , Ric1-Rgp1 complex was purified from insect cells with anti-FLAG antibody-agarose followed by immunoblotting with anti-FLAG and anti-Myc antibodies to detect Rgp1 and
Figure Legend Snippet: Ric1-Rgp1 complex has guanine nucleotide exchange activity toward Rab6A in vitro . A , Ric1-Rgp1 complex was purified from insect cells with anti-FLAG antibody-agarose followed by immunoblotting with anti-FLAG and anti-Myc antibodies to detect Rgp1 and

Techniques Used: Activity Assay, In Vitro, Purification

Ric1 is an effector of Rab33B. A , HEK293T cells were co-transfected with either GFP-Rab4A or GFP-Rab33B and FLAG-Rgp1 and Myc-Ric1 for 24 h. GFP-Rab4A and GFP-Rab33B were immunoprecipitated ( IP ) with GFP-binding protein-agarose followed by immunoblotting
Figure Legend Snippet: Ric1 is an effector of Rab33B. A , HEK293T cells were co-transfected with either GFP-Rab4A or GFP-Rab33B and FLAG-Rgp1 and Myc-Ric1 for 24 h. GFP-Rab4A and GFP-Rab33B were immunoprecipitated ( IP ) with GFP-binding protein-agarose followed by immunoblotting

Techniques Used: Transfection, Immunoprecipitation, Binding Assay

24) Product Images from "Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners *"

Article Title: Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.614107

Evidence that interaction of the PspC C-terminal domain with PspA or PspB is mutually exclusive in vitro . A , summary of the protocol used for the GST fusion protein two-phase membrane lysate pulldown assay. B , immunoblot analysis. In Experiment A , GST or GST fused to the PspC C-terminal domain ( GST-PspC CT ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspA ( prey lysate 1 ). After washing, proteins were recovered from half of the beads by boiling in SDS-PAGE sample buffer ( Elution 1 ). The other half of the beads was incubated with a second detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspB ( prey lysate 2 ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer ( Elution 2 ). Experiment B was done similarly, except that the order of incubation with the PspA and PspB membrane lysates was reversed. For each experiment, membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane (for experiments A and B the elution samples for Ponceau S staining were run on the same gels, but irrelevant lanes between elutions 1 and 2 have been removed).
Figure Legend Snippet: Evidence that interaction of the PspC C-terminal domain with PspA or PspB is mutually exclusive in vitro . A , summary of the protocol used for the GST fusion protein two-phase membrane lysate pulldown assay. B , immunoblot analysis. In Experiment A , GST or GST fused to the PspC C-terminal domain ( GST-PspC CT ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspA ( prey lysate 1 ). After washing, proteins were recovered from half of the beads by boiling in SDS-PAGE sample buffer ( Elution 1 ). The other half of the beads was incubated with a second detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspB ( prey lysate 2 ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer ( Elution 2 ). Experiment B was done similarly, except that the order of incubation with the PspA and PspB membrane lysates was reversed. For each experiment, membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane (for experiments A and B the elution samples for Ponceau S staining were run on the same gels, but irrelevant lanes between elutions 1 and 2 have been removed).

Techniques Used: In Vitro, Incubation, SDS Page, Staining

GST-PspC CT fusion protein pulldown assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain with all core Psp proteins (Psp + ) or in which the only core Psp proteins present were PspA or PspB as indicated ( Prey ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane.
Figure Legend Snippet: GST-PspC CT fusion protein pulldown assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain with all core Psp proteins (Psp + ) or in which the only core Psp proteins present were PspA or PspB as indicated ( Prey ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane.

Techniques Used: Mutagenesis, Incubation, SDS Page, Staining

In vitro GST/MBP fusion protein interaction assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) were bound to glutathione-Sepharose (beads) and incubated with 15 μg of MBP fused to the C-terminal domain of PspB ( MBP-PspB CT ) or to LacZα ( MBP-LacZ α). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Samples of each purified MBP-fusion protein ( Inputs ) and the recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with anti-MBP or anti-GST monoclonal antibodies. MBP-LacZα underwent apparent degradation during purification, leading to the isolation of both full-length and truncated protein.
Figure Legend Snippet: In vitro GST/MBP fusion protein interaction assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) were bound to glutathione-Sepharose (beads) and incubated with 15 μg of MBP fused to the C-terminal domain of PspB ( MBP-PspB CT ) or to LacZα ( MBP-LacZ α). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Samples of each purified MBP-fusion protein ( Inputs ) and the recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with anti-MBP or anti-GST monoclonal antibodies. MBP-LacZα underwent apparent degradation during purification, leading to the isolation of both full-length and truncated protein.

Techniques Used: In Vitro, Protein Interaction Assay, Mutagenesis, Incubation, SDS Page, Purification, Isolation

25) Product Images from "GLUT4 Enhancer Factor (GEF) Interacts with MEF2A and HDAC5 to Regulate the GLUT4 Promoter in Adipocytes *"

Article Title: GLUT4 Enhancer Factor (GEF) Interacts with MEF2A and HDAC5 to Regulate the GLUT4 Promoter in Adipocytes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M800481200

Homo-oligomerization of GEF. GST-GEF can homo-oligomerize, but not within the DNA-binding domain ( DB ). A , schematic representation of GST- and HA-tagged GEF utilized in co-precipitations. GEF-11 comprises residues 68–387 of GEF-FL. B , nuclear extracts from COS-7 cells transiently expressing HA-GEF-11 were incubated with various GST-GEF fusion proteins bound to glutathione-Sepharose beads. Protein complexes were washed, eluted by denaturation, and analyzed by SDS-PAGE and immunoblotting for HA. FL , full-length.
Figure Legend Snippet: Homo-oligomerization of GEF. GST-GEF can homo-oligomerize, but not within the DNA-binding domain ( DB ). A , schematic representation of GST- and HA-tagged GEF utilized in co-precipitations. GEF-11 comprises residues 68–387 of GEF-FL. B , nuclear extracts from COS-7 cells transiently expressing HA-GEF-11 were incubated with various GST-GEF fusion proteins bound to glutathione-Sepharose beads. Protein complexes were washed, eluted by denaturation, and analyzed by SDS-PAGE and immunoblotting for HA. FL , full-length.

Techniques Used: Binding Assay, Expressing, Incubation, SDS Page

GEF can interact with HDAC5. A , cDNA from a portion of HDAC5 was expanded using RT-PCR from mouse adipose ( A ) or skeletal muscle ( S ) RNA and specific primers corresponding to an approximate 500-bp region of HDAC5. B , whole cell extracts from COS-7 cells transiently expressing HDAC were incubated with bacterially expressed GST alone or GST-GEF-FL bound to glutathione-Sepharose. Protein complexes were washed, eluted by denaturation, and analyzed by SDS-PAGE and immunoblotting ( IB ) for HDAC5. C , proteins from COS-7 whole cell extracts from cells overexpressing GEF alone, HDAC5 alone, or GEF and HDAC5 together were immunoprecipitated ( IP ) using affinity purified anti-GEF polyclonal antibodies and Protein A/G PLUS-agarose. Immunoprecipitates were eluted by denaturation and analyzed by SDS-PAGE and immunoblot for HDAC5 ( lanes 1–3 ). Input (3%) was loaded as a comparison for HDAC levels ( lanes 4–6 ) or GEF ( lanes 7–9 ). FL , full-length.
Figure Legend Snippet: GEF can interact with HDAC5. A , cDNA from a portion of HDAC5 was expanded using RT-PCR from mouse adipose ( A ) or skeletal muscle ( S ) RNA and specific primers corresponding to an approximate 500-bp region of HDAC5. B , whole cell extracts from COS-7 cells transiently expressing HDAC were incubated with bacterially expressed GST alone or GST-GEF-FL bound to glutathione-Sepharose. Protein complexes were washed, eluted by denaturation, and analyzed by SDS-PAGE and immunoblotting ( IB ) for HDAC5. C , proteins from COS-7 whole cell extracts from cells overexpressing GEF alone, HDAC5 alone, or GEF and HDAC5 together were immunoprecipitated ( IP ) using affinity purified anti-GEF polyclonal antibodies and Protein A/G PLUS-agarose. Immunoprecipitates were eluted by denaturation and analyzed by SDS-PAGE and immunoblot for HDAC5 ( lanes 1–3 ). Input (3%) was loaded as a comparison for HDAC levels ( lanes 4–6 ) or GEF ( lanes 7–9 ). FL , full-length.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, SDS Page, Immunoprecipitation, Affinity Purification

GEF can increase the association of HDAC5 with FLAG-MEF2D. A , whole cell extracts of COS-7 transiently transfected with plasmids encoding FLAG-MEF2D, HDAC5, and increasing amounts of GEF were co-immunoprecipitated ( IP ) with anti-FLAG-agarose. Immunoprecipitated HDAC5 and GEF ( lanes 1–3 ) were analyzed by SDS-PAGE and immunoblot ( IB ) and compared with 3% total input ( lanes 4–6 ). B , the ratio of intensities of the HDAC5 bands from IP and input lanes were calculated in the absence (0 mg, lanes 1 and 4 ) and presence (1.5 mg, lanes 2 and 5 ; 3 mg, lanes 3 and 6 ) of GEF. The -fold increase of the HDAC5 IP/input ratio from 0 mg of GEF was statistically significant when comparing 0 to 3 mg of GEF ( p
Figure Legend Snippet: GEF can increase the association of HDAC5 with FLAG-MEF2D. A , whole cell extracts of COS-7 transiently transfected with plasmids encoding FLAG-MEF2D, HDAC5, and increasing amounts of GEF were co-immunoprecipitated ( IP ) with anti-FLAG-agarose. Immunoprecipitated HDAC5 and GEF ( lanes 1–3 ) were analyzed by SDS-PAGE and immunoblot ( IB ) and compared with 3% total input ( lanes 4–6 ). B , the ratio of intensities of the HDAC5 bands from IP and input lanes were calculated in the absence (0 mg, lanes 1 and 4 ) and presence (1.5 mg, lanes 2 and 5 ; 3 mg, lanes 3 and 6 ) of GEF. The -fold increase of the HDAC5 IP/input ratio from 0 mg of GEF was statistically significant when comparing 0 to 3 mg of GEF ( p

Techniques Used: Transfection, Immunoprecipitation, SDS Page

26) Product Images from "Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor Kinase"

Article Title: Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor Kinase

Journal: Plant Physiology

doi: 10.1104/pp.103.023846

Calmodulin binds to the kinase domain of SRK in vitro. A, Binding of GST or the GST protein fused with either wild-type or mutant forms of the SRK 29 kinase domain to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. B, Binding of the integral SRK protein to calmodulin-Sepharose beads. A recombinant hexa-His epitope-tagged, kinase-inactive form of SRK ( m SRK 3 ). C, Binding of Brassica calmodulin to glutathione-Sepharose beads carrying either GST or GST protein fused to either wild-type or mutant forms of the SRK 29 kinase domain. Eluted proteins were detected by silver staining. Note that calmodulin alters its conformation in the presence of calcium resulting in a change in mobility. CAM, Calmodulin with no bound Ca 2+ ; CAM/Ca, calmodulin with bound Ca 2+ ; C, purified fusion protein; UB, unbound fraction; W1 and W40, washes; E1 to E4, eluted fractions. C and UB, One-quarter of the amount of protein loaded on and unbound to the column, respectively.
Figure Legend Snippet: Calmodulin binds to the kinase domain of SRK in vitro. A, Binding of GST or the GST protein fused with either wild-type or mutant forms of the SRK 29 kinase domain to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. B, Binding of the integral SRK protein to calmodulin-Sepharose beads. A recombinant hexa-His epitope-tagged, kinase-inactive form of SRK ( m SRK 3 ). C, Binding of Brassica calmodulin to glutathione-Sepharose beads carrying either GST or GST protein fused to either wild-type or mutant forms of the SRK 29 kinase domain. Eluted proteins were detected by silver staining. Note that calmodulin alters its conformation in the presence of calcium resulting in a change in mobility. CAM, Calmodulin with no bound Ca 2+ ; CAM/Ca, calmodulin with bound Ca 2+ ; C, purified fusion protein; UB, unbound fraction; W1 and W40, washes; E1 to E4, eluted fractions. C and UB, One-quarter of the amount of protein loaded on and unbound to the column, respectively.

Techniques Used: In Vitro, Binding Assay, Mutagenesis, Recombinant, Silver Staining, Chick Chorioallantoic Membrane Assay, Purification

Calmodulin binds to sub-domain VIa of the kinase domain of SRK in vitro. A, Amino acid sequence of the HEL1 region of sub-domain VIa of SRK 3 . The wheel projection shows the amphiphilic nature of HEL1. The sequence of the control peptide PEP1 is also shown. B, Binding of GST::HEL1 and GST::PEP1 fusion proteins to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. C, purified GST fusion protein; UB, unbound fraction; W1 and W40, first and last washes; E1 to E4, eluted fractions.
Figure Legend Snippet: Calmodulin binds to sub-domain VIa of the kinase domain of SRK in vitro. A, Amino acid sequence of the HEL1 region of sub-domain VIa of SRK 3 . The wheel projection shows the amphiphilic nature of HEL1. The sequence of the control peptide PEP1 is also shown. B, Binding of GST::HEL1 and GST::PEP1 fusion proteins to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. C, purified GST fusion protein; UB, unbound fraction; W1 and W40, first and last washes; E1 to E4, eluted fractions.

Techniques Used: In Vitro, Sequencing, Binding Assay, Purification

Calmodulin binds to the kinase domains of SFR1, RLK4, and CLV1, but not BRI1, in vitro. A, Analysis of the binding of the kinase domains of SFR1, RLK4, CLV1, and BRI1 to calmodulin-Sepharose. The kinase domains were expressed as fusion proteins with either GST or MBP. Both a wild-type (GST::SFR1kin) and a kinase-inactive mutant form (GST::SFR1kin K555R ) of the SFR1 kinase domain were tested. GST- and MBP-containing proteins were detected with anti-GST and anti-MBP antibodies, respectively. B, Abundance of calmodulin transcripts in different Brassica organs. Calmodulin transcripts were detected with a probe corresponding to the entire coding sequence. Lower, Ethidium bromide-stained rRNA. The positions of RNA size markers are shown at right in kilobase pairs. R, Root; C, cotyledon; L, leaf; Se, sepal; P, petal; A, anther; O, ovary; St, stigma.
Figure Legend Snippet: Calmodulin binds to the kinase domains of SFR1, RLK4, and CLV1, but not BRI1, in vitro. A, Analysis of the binding of the kinase domains of SFR1, RLK4, CLV1, and BRI1 to calmodulin-Sepharose. The kinase domains were expressed as fusion proteins with either GST or MBP. Both a wild-type (GST::SFR1kin) and a kinase-inactive mutant form (GST::SFR1kin K555R ) of the SFR1 kinase domain were tested. GST- and MBP-containing proteins were detected with anti-GST and anti-MBP antibodies, respectively. B, Abundance of calmodulin transcripts in different Brassica organs. Calmodulin transcripts were detected with a probe corresponding to the entire coding sequence. Lower, Ethidium bromide-stained rRNA. The positions of RNA size markers are shown at right in kilobase pairs. R, Root; C, cotyledon; L, leaf; Se, sepal; P, petal; A, anther; O, ovary; St, stigma.

Techniques Used: In Vitro, Binding Assay, Mutagenesis, Sequencing, Staining

27) Product Images from "RAM function is dependent on Kap?2-mediated nuclear entry"

Article Title: RAM function is dependent on Kap?2-mediated nuclear entry

Journal: Biochemical Journal

doi: 10.1042/BJ20131359

Kapβ2 mediates RAM import ( A ) Recombinant GST–RAM or GST was incubated with recombinant Kapβ2. GST–RAM and Kapβ2 complexes were purified on glutathione–Sepharose, resolved by SDS/PAGE and visualized by Western blotting. Molecular masses are indicated in kDa. ( B ) HeLa cells were transfected with pcDNA5 Myc-Kapβ2 or vector control and pcDNA5 RAM-GFP or GFP. Immunoprecipitations (IP) were performed on cell extracts using anti-Myc antibodies. Western blots were performed to detect Myc–Kapβ2, RAM and β-tubulin in inputs and immunoprecipitates. ( C ) HeLa cells were transfected with pcDNA5 Myc-Kapβ2 or vector control (c) and pcDNA5 RAM-GFP, RAM-GFP PY/AA mutants or GFP. Immunoprecipitations were performed on cell extracts using anti-Myc antibodies. Western blots were performed to detect Myc–Kapβ2, RAM and β-tubulin in inputs and immunoprecipitates. ( D ) HeLa cells were transfected with two independent Kapβ2 siRNAs or control siRNA. After 2 days RNA was extracted and real-time PCR performed to detect expression of Kapβ2, RNMT and RAM. The average result for three independent experiments is presented and the error bars indicate±S.D. ( E ) Western blots were performed to detect Kapβ2, RNMT, RAM and β-tubulin in cell extracts. ( F ) IF was used to detect RAM localization and DAPI staining was used to detect nuclei. The overlay of RAM IF, DAPI staining and bright field is also presented. si, siRNA. ( G ) HeLa cells were transfected with pcDNA3.1 Myc-M9M or vector control. IF was used to detect RAM and Myc-M9M. DAPI staining was used to detect nuclei. The overlay of RAM or RNMT, DAPI staining and bright field is presented.
Figure Legend Snippet: Kapβ2 mediates RAM import ( A ) Recombinant GST–RAM or GST was incubated with recombinant Kapβ2. GST–RAM and Kapβ2 complexes were purified on glutathione–Sepharose, resolved by SDS/PAGE and visualized by Western blotting. Molecular masses are indicated in kDa. ( B ) HeLa cells were transfected with pcDNA5 Myc-Kapβ2 or vector control and pcDNA5 RAM-GFP or GFP. Immunoprecipitations (IP) were performed on cell extracts using anti-Myc antibodies. Western blots were performed to detect Myc–Kapβ2, RAM and β-tubulin in inputs and immunoprecipitates. ( C ) HeLa cells were transfected with pcDNA5 Myc-Kapβ2 or vector control (c) and pcDNA5 RAM-GFP, RAM-GFP PY/AA mutants or GFP. Immunoprecipitations were performed on cell extracts using anti-Myc antibodies. Western blots were performed to detect Myc–Kapβ2, RAM and β-tubulin in inputs and immunoprecipitates. ( D ) HeLa cells were transfected with two independent Kapβ2 siRNAs or control siRNA. After 2 days RNA was extracted and real-time PCR performed to detect expression of Kapβ2, RNMT and RAM. The average result for three independent experiments is presented and the error bars indicate±S.D. ( E ) Western blots were performed to detect Kapβ2, RNMT, RAM and β-tubulin in cell extracts. ( F ) IF was used to detect RAM localization and DAPI staining was used to detect nuclei. The overlay of RAM IF, DAPI staining and bright field is also presented. si, siRNA. ( G ) HeLa cells were transfected with pcDNA3.1 Myc-M9M or vector control. IF was used to detect RAM and Myc-M9M. DAPI staining was used to detect nuclei. The overlay of RAM or RNMT, DAPI staining and bright field is presented.

Techniques Used: Recombinant, Incubation, Purification, SDS Page, Western Blot, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Staining

RAM 1–45 activates RNMT ( A ) Recombinant GST–RAM WT, truncation mutants or GST alone were incubated with recombinant RNMT. GST–RAM complexes were purified on glutathione–Sepharose, resolved by SDS/PAGE and co-purified RNMT was visualised by Coomassie Blue staining and Western blotting (WB). Molecular masses are indicated in kDa. ( B ) HeLa cells were transfected with pcDNA5 HA-RNMT or pcDNA5 (c), and pcDNA4 RAM-GFP (RAM WT), RAM-GFP mutants or GFP. Immunoprecipitations (IP) were performed with anti-HA and anti-GFP antibodies. Western blots were performed to detect GFP, RAM, HA and RNMT in inputs and immunoprecipitates. (* indicates cross-reacting antibody heavy or light chain). ( C ) Cap methyltransferase assay was performed using 15 nM RNMT plus 15 nM GST–RAM, truncation mutants or GST control. Protein complexes were incubated with [ 32 P]GpppG transcript and S -adenosylmethionine for 10 mins. Following the reaction, transcripts were digested and GpppG and m7GpppG were resolved by TLC and visualized by phosphoimaging. A representative image is shown. The average fold change in cap methyltransferase activity compared with that generated by RNMT alone for six independent experiments is depicted. Error bars indicate±S.D. *** P
Figure Legend Snippet: RAM 1–45 activates RNMT ( A ) Recombinant GST–RAM WT, truncation mutants or GST alone were incubated with recombinant RNMT. GST–RAM complexes were purified on glutathione–Sepharose, resolved by SDS/PAGE and co-purified RNMT was visualised by Coomassie Blue staining and Western blotting (WB). Molecular masses are indicated in kDa. ( B ) HeLa cells were transfected with pcDNA5 HA-RNMT or pcDNA5 (c), and pcDNA4 RAM-GFP (RAM WT), RAM-GFP mutants or GFP. Immunoprecipitations (IP) were performed with anti-HA and anti-GFP antibodies. Western blots were performed to detect GFP, RAM, HA and RNMT in inputs and immunoprecipitates. (* indicates cross-reacting antibody heavy or light chain). ( C ) Cap methyltransferase assay was performed using 15 nM RNMT plus 15 nM GST–RAM, truncation mutants or GST control. Protein complexes were incubated with [ 32 P]GpppG transcript and S -adenosylmethionine for 10 mins. Following the reaction, transcripts were digested and GpppG and m7GpppG were resolved by TLC and visualized by phosphoimaging. A representative image is shown. The average fold change in cap methyltransferase activity compared with that generated by RNMT alone for six independent experiments is depicted. Error bars indicate±S.D. *** P

Techniques Used: Recombinant, Incubation, Purification, SDS Page, Staining, Western Blot, Transfection, Thin Layer Chromatography, Activity Assay, Generated

RAM nuclear localization is dependent on the C-terminus ( A ) HeLa cells were transfected with pcDNA5 Fg-RAM WT, truncation mutants or vector control. Immunoprecipitations (IP) were performed on normalized cell extracts using anti-Fg antibody–agarose conjugates. Western blots were performed to detect Fg-tagged proteins, RNMT and β-tubulin in immunoprecipitates and extracts. Molecular masses are indicated in kDa. ( B ) HeLa cells were transfected with control or RAM siRNA and with pcDNA5 Fg-RAM WT, truncation mutants or vector control. IF analysis was used to detect RAM localization and DAPI staining was used to detect nuclei. The overlay of RAM IF, DAPI staining and bright field is also presented.
Figure Legend Snippet: RAM nuclear localization is dependent on the C-terminus ( A ) HeLa cells were transfected with pcDNA5 Fg-RAM WT, truncation mutants or vector control. Immunoprecipitations (IP) were performed on normalized cell extracts using anti-Fg antibody–agarose conjugates. Western blots were performed to detect Fg-tagged proteins, RNMT and β-tubulin in immunoprecipitates and extracts. Molecular masses are indicated in kDa. ( B ) HeLa cells were transfected with control or RAM siRNA and with pcDNA5 Fg-RAM WT, truncation mutants or vector control. IF analysis was used to detect RAM localization and DAPI staining was used to detect nuclei. The overlay of RAM IF, DAPI staining and bright field is also presented.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Staining

28) Product Images from "Dimerization of Sterol Regulatory Element-Binding Protein 2 via the Helix-Loop-Helix-Leucine Zipper Domain Is a Prerequisite for Its Nuclear Localization Mediated by Importin ?"

Article Title: Dimerization of Sterol Regulatory Element-Binding Protein 2 via the Helix-Loop-Helix-Leucine Zipper Domain Is a Prerequisite for Its Nuclear Localization Mediated by Importin ?

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.8.2779-2789.2001

Mapping of the SREBP-2 binding domain of importin β. (A) Full-length (1 to 876) and various truncated mutants of importin β were produced as GST fusion proteins and tested for their ability to bind to SREBP-2. Purified GST or GST-importin β fragments (150 pmol) were incubated with 270 μl of E. coli lysate expressing His-SREBP-2 (300 μl final volume). GST fusion proteins were then absorbed to 15 μl of glutathione-Sepharose beads. After extensive washing, the bound proteins were eluted by boiling in SDS-PAGE sample buffer and divided into two equal portions. Each portion was separated by SDS–10% PAGE and analyzed by immunoblotting using the monoclonal anti-penta-His antibody (top) or by Coomassie staining (bottom). E. coli lysate (13 μl) expressing His-tagged SREBP-2 was directly applied to each gel (10% input). Asterisks indicate the positions of the GST and GST-importin β fragments which were absorbed to the glutathione-Sepharose beads (bottom). (B) Schematic representation of the importin β deletion mutants used in this study. All mutants were expressed as GST fusion proteins. Numbers indicate the amino acid position of each importin β fragment.
Figure Legend Snippet: Mapping of the SREBP-2 binding domain of importin β. (A) Full-length (1 to 876) and various truncated mutants of importin β were produced as GST fusion proteins and tested for their ability to bind to SREBP-2. Purified GST or GST-importin β fragments (150 pmol) were incubated with 270 μl of E. coli lysate expressing His-SREBP-2 (300 μl final volume). GST fusion proteins were then absorbed to 15 μl of glutathione-Sepharose beads. After extensive washing, the bound proteins were eluted by boiling in SDS-PAGE sample buffer and divided into two equal portions. Each portion was separated by SDS–10% PAGE and analyzed by immunoblotting using the monoclonal anti-penta-His antibody (top) or by Coomassie staining (bottom). E. coli lysate (13 μl) expressing His-tagged SREBP-2 was directly applied to each gel (10% input). Asterisks indicate the positions of the GST and GST-importin β fragments which were absorbed to the glutathione-Sepharose beads (bottom). (B) Schematic representation of the importin β deletion mutants used in this study. All mutants were expressed as GST fusion proteins. Numbers indicate the amino acid position of each importin β fragment.

Techniques Used: Binding Assay, Produced, Purification, Incubation, Expressing, SDS Page, Polyacrylamide Gel Electrophoresis, Staining

IBB competes with SREBP-2 for binding to importin β and nuclear import. (A) The IBB domain competes for binding of HLH-Zip of SREBP-2 to importin β. GST or GST-importin β (GST-β) (70 pmol) was incubated with 70 pmol of GFP-SREBP-2(343–403) (GFP-HLHZ) in the presence or absence of MBP-IBB (70 pmol). The GST fusion proteins were then recovered on glutathione-Sepharose beads, and the bound proteins were analyzed by SDS–10% PAGE followed by Coomassie staining. (B) Competition with nuclear import by the IBB domain. Nuclear import of GST-NLS-GFP and Flag-SREBP-2 in digitonin-permeabilized HeLa cells was performed by incubating the cells with 10 μl of reaction mixture containing 4 pmol of GST-NLS-GFP or Flag-SREBP-2 in the presence of cytosol from Ehrlich tumor cells and an ATP regeneration system (left panels). For competition by IBB, import assays were carried out in the presence of 120 pmol of MBP-IBB (right panels).
Figure Legend Snippet: IBB competes with SREBP-2 for binding to importin β and nuclear import. (A) The IBB domain competes for binding of HLH-Zip of SREBP-2 to importin β. GST or GST-importin β (GST-β) (70 pmol) was incubated with 70 pmol of GFP-SREBP-2(343–403) (GFP-HLHZ) in the presence or absence of MBP-IBB (70 pmol). The GST fusion proteins were then recovered on glutathione-Sepharose beads, and the bound proteins were analyzed by SDS–10% PAGE followed by Coomassie staining. (B) Competition with nuclear import by the IBB domain. Nuclear import of GST-NLS-GFP and Flag-SREBP-2 in digitonin-permeabilized HeLa cells was performed by incubating the cells with 10 μl of reaction mixture containing 4 pmol of GST-NLS-GFP or Flag-SREBP-2 in the presence of cytosol from Ehrlich tumor cells and an ATP regeneration system (left panels). For competition by IBB, import assays were carried out in the presence of 120 pmol of MBP-IBB (right panels).

Techniques Used: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Staining

SREBP-2/L1.2.3A mutant has a reduced affinity for importin β. (A) Purified Flag-SREBP-2 (WT) or Flag-SREBP-2/L1.2.3.A (25 nM or 1.6 μM in a total volume of 50 μl) was incubated with GST or GST-importin β (1 μM) for 20 min at room temperature in the presence of 5 μl of glutathione-Sepharose beads. After incubation, the beads were collected by centrifugation followed by extensive washing, and the bound proteins were eluted by boiling in SDS-PAGE sample buffer. Proteins were separated SDS–10% PAGE and analyzed by immunoblotting with anti-Flag M2 monoclonal antibody and anti-GST polyclonal antibodies. Control samples of recombinant Flag-SREBP-2 and Flag-SREBP-2/L1.2.3A (10 ng each) were loaded directly onto the gel. (B) The in vitro-translated L1.2.3A mutant does not bind to immobilized GST-importin β in the reticulocyte lysate. 35 S-labeled wild-type (WT) and L1.2.3A mutant SREBP-2 were translated in vitro, and 5 μl of each was incubated with 60 pmol each of GST or GST-importin β in 50 μl of binding buffer C. GST proteins were then absorbed to 5 μl of glutathione-Sepharose beads. After washing three times, the bound proteins were eluted by boiling the beads in SDS-PAGE sample buffer. Half of each eluate was separated on SDS–10% PAGE and analyzed by autoradiography. 35 S-labeled SREBP-2 and SREBP-2/L1.2.3A (2.5 μl each) were loaded directly as a control (input).
Figure Legend Snippet: SREBP-2/L1.2.3A mutant has a reduced affinity for importin β. (A) Purified Flag-SREBP-2 (WT) or Flag-SREBP-2/L1.2.3.A (25 nM or 1.6 μM in a total volume of 50 μl) was incubated with GST or GST-importin β (1 μM) for 20 min at room temperature in the presence of 5 μl of glutathione-Sepharose beads. After incubation, the beads were collected by centrifugation followed by extensive washing, and the bound proteins were eluted by boiling in SDS-PAGE sample buffer. Proteins were separated SDS–10% PAGE and analyzed by immunoblotting with anti-Flag M2 monoclonal antibody and anti-GST polyclonal antibodies. Control samples of recombinant Flag-SREBP-2 and Flag-SREBP-2/L1.2.3A (10 ng each) were loaded directly onto the gel. (B) The in vitro-translated L1.2.3A mutant does not bind to immobilized GST-importin β in the reticulocyte lysate. 35 S-labeled wild-type (WT) and L1.2.3A mutant SREBP-2 were translated in vitro, and 5 μl of each was incubated with 60 pmol each of GST or GST-importin β in 50 μl of binding buffer C. GST proteins were then absorbed to 5 μl of glutathione-Sepharose beads. After washing three times, the bound proteins were eluted by boiling the beads in SDS-PAGE sample buffer. Half of each eluate was separated on SDS–10% PAGE and analyzed by autoradiography. 35 S-labeled SREBP-2 and SREBP-2/L1.2.3A (2.5 μl each) were loaded directly as a control (input).

Techniques Used: Mutagenesis, Purification, Incubation, Centrifugation, SDS Page, Polyacrylamide Gel Electrophoresis, Recombinant, In Vitro, Labeling, Binding Assay, Autoradiography

29) Product Images from "Human Telomeres Maintain Their Overhang Length at Senescence †"

Article Title: Human Telomeres Maintain Their Overhang Length at Senescence †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.6.2158-2168.2005

IMR90 cells maintain their telomeric 3′ overhangs at senescence. (A) Overhang protection analysis of DNA from IMR90 fetal lung fibroblasts at different PD up to senescence (sen) and from IMR90 cells expressing SV40 L-Tg or HPV E6 and E7 (E6/E7). Cells that are in the extended life span through expression of L-Tg (PD 74) or E6/E7 (PD 87) are indicated as M1-M2. DNA was treated with or without Exo I before being analyzed in the overhang protection assay. To verify approximately equal amounts of input DNA, 1/30 of each sample was run on a 0.7% agarose gel and visualized with ethidium bromide (input). (B) Weighted mean sizes of overhangs were quantified and plotted. Results shown are representative of at least three independent experiments. Error bars represent one standard deviation. (C) Nondenaturing hybridization analysis of telomeric restriction fragments from young (PD 30) and senescent (PD 58) IMR90 fetal lung fibroblasts. Undigested genomic DNA was hybridized to 32 P-labeled C-rich high-specificity probe and then gel fractionated in 0.5× TBE. After the gel was denatured, overhang signals were transferred to a nylon membrane and exposed to a phosphorimager screen (left panel). The denatured gel was then neutralized and rehybridized to the Alu repeat probe (right panel). (D) Relative amounts of overhangs were calculated by normalizing signals from the membrane (overhang signals) to the Alu repeat signal (representing total genomic DNA) and plotted. Results shown are representative of three independent experiments. Error bars represent one standard deviation.
Figure Legend Snippet: IMR90 cells maintain their telomeric 3′ overhangs at senescence. (A) Overhang protection analysis of DNA from IMR90 fetal lung fibroblasts at different PD up to senescence (sen) and from IMR90 cells expressing SV40 L-Tg or HPV E6 and E7 (E6/E7). Cells that are in the extended life span through expression of L-Tg (PD 74) or E6/E7 (PD 87) are indicated as M1-M2. DNA was treated with or without Exo I before being analyzed in the overhang protection assay. To verify approximately equal amounts of input DNA, 1/30 of each sample was run on a 0.7% agarose gel and visualized with ethidium bromide (input). (B) Weighted mean sizes of overhangs were quantified and plotted. Results shown are representative of at least three independent experiments. Error bars represent one standard deviation. (C) Nondenaturing hybridization analysis of telomeric restriction fragments from young (PD 30) and senescent (PD 58) IMR90 fetal lung fibroblasts. Undigested genomic DNA was hybridized to 32 P-labeled C-rich high-specificity probe and then gel fractionated in 0.5× TBE. After the gel was denatured, overhang signals were transferred to a nylon membrane and exposed to a phosphorimager screen (left panel). The denatured gel was then neutralized and rehybridized to the Alu repeat probe (right panel). (D) Relative amounts of overhangs were calculated by normalizing signals from the membrane (overhang signals) to the Alu repeat signal (representing total genomic DNA) and plotted. Results shown are representative of three independent experiments. Error bars represent one standard deviation.

Techniques Used: Expressing, Agarose Gel Electrophoresis, Standard Deviation, Hybridization, Labeling

BJ cells maintain their telomeric 3′ overhangs at senescence. (A) Overhang protection assay of DNA from BJ fibroblasts at different PD up to senescence (sen). DNA was treated with or without Exo I before being analyzed in the overhang protection assay. To verify approximately equal amounts of input DNA, 1/30 of each sample was run on a 0.7% agarose gel and visualized with ethidium bromide (input). (B) Weighted mean sizes of the overhangs were quantified and are plotted. Results shown are representative of at least three independent experiments. Error bars represent one standard deviation. Cells were cultured in three different growth series until senescence, and DNA from these cells was used for the overhang protection assay. (C) Senescence-associated β-galactosidase staining of BJ fibroblasts at PD 32 and at senescence. (D) Nondenaturing hybridization analysis of telomeric restriction fragments from BJ fibroblasts at PD 40 and 90 (senescence). Undigested genomic DNA was hybridized to 32 P-labeled C-rich high-specificity probe and then gel fractionated in 0.5× TBE. After the gel was denatured, overhang signals were transferred to a nylon membrane and exposed to a phosphorimager screen (left panel). The denatured gel was neutralized and rehybridized to the Alu repeat probe (right panel). (E) Relative amounts of overhangs were calculated by normalizing signals from the membrane (overhang signals) by the Alu repeat signal (representing total genomic DNA) and plotted. Results shown are representative of three independent experiments. Error bars represent one standard deviation.
Figure Legend Snippet: BJ cells maintain their telomeric 3′ overhangs at senescence. (A) Overhang protection assay of DNA from BJ fibroblasts at different PD up to senescence (sen). DNA was treated with or without Exo I before being analyzed in the overhang protection assay. To verify approximately equal amounts of input DNA, 1/30 of each sample was run on a 0.7% agarose gel and visualized with ethidium bromide (input). (B) Weighted mean sizes of the overhangs were quantified and are plotted. Results shown are representative of at least three independent experiments. Error bars represent one standard deviation. Cells were cultured in three different growth series until senescence, and DNA from these cells was used for the overhang protection assay. (C) Senescence-associated β-galactosidase staining of BJ fibroblasts at PD 32 and at senescence. (D) Nondenaturing hybridization analysis of telomeric restriction fragments from BJ fibroblasts at PD 40 and 90 (senescence). Undigested genomic DNA was hybridized to 32 P-labeled C-rich high-specificity probe and then gel fractionated in 0.5× TBE. After the gel was denatured, overhang signals were transferred to a nylon membrane and exposed to a phosphorimager screen (left panel). The denatured gel was neutralized and rehybridized to the Alu repeat probe (right panel). (E) Relative amounts of overhangs were calculated by normalizing signals from the membrane (overhang signals) by the Alu repeat signal (representing total genomic DNA) and plotted. Results shown are representative of three independent experiments. Error bars represent one standard deviation.

Techniques Used: Agarose Gel Electrophoresis, Standard Deviation, Cell Culture, Staining, Hybridization, Labeling

Loss of telomeric overhangs in cells expressing SV40 L-Tg, as shown in the overhang protection analysis of DNA from BJ fibroblasts infected with retrovirus containing SV40 L-Tg at different PD. Cells bypassed senescence at approximately PD 90. DNA was treated with or without Exo I before being analyzed in the overhang protection assay. To verify approximately equal amounts of input DNA, 1/30 of each sample was run on a 0.7% agarose gel and visualized with ethidium bromide (input). (B) Weighted mean sizes of overhangs were quantified and are plotted. Results shown are representative of at least three independent experiments. Error bars represent one standard deviation. (C) Relative amount of overhangs of BJ/L-Tg cells measured by nondenaturing hybridization assay. Undigested whole genomic DNA from BJ at PD 40 and at senescence was first hybridized to the C-rich telomeric probe and then gel fractionated. After the gel was denatured, overhang signals were transferred to a membrane and the gel was rehybridized to the Alu repeat probe. Relative amounts of overhangs were calculated by normalizing signals from the membrane (overhang signals) by the Alu repeat signal (representing total genomic DNA). Results shown are representative of three independent experiments. Error bars represent one standard deviation.
Figure Legend Snippet: Loss of telomeric overhangs in cells expressing SV40 L-Tg, as shown in the overhang protection analysis of DNA from BJ fibroblasts infected with retrovirus containing SV40 L-Tg at different PD. Cells bypassed senescence at approximately PD 90. DNA was treated with or without Exo I before being analyzed in the overhang protection assay. To verify approximately equal amounts of input DNA, 1/30 of each sample was run on a 0.7% agarose gel and visualized with ethidium bromide (input). (B) Weighted mean sizes of overhangs were quantified and are plotted. Results shown are representative of at least three independent experiments. Error bars represent one standard deviation. (C) Relative amount of overhangs of BJ/L-Tg cells measured by nondenaturing hybridization assay. Undigested whole genomic DNA from BJ at PD 40 and at senescence was first hybridized to the C-rich telomeric probe and then gel fractionated. After the gel was denatured, overhang signals were transferred to a membrane and the gel was rehybridized to the Alu repeat probe. Relative amounts of overhangs were calculated by normalizing signals from the membrane (overhang signals) by the Alu repeat signal (representing total genomic DNA). Results shown are representative of three independent experiments. Error bars represent one standard deviation.

Techniques Used: Expressing, Infection, Agarose Gel Electrophoresis, Standard Deviation, Hybridization

30) Product Images from "Scapinin, the Protein Phosphatase 1 Binding Protein, Enhances Cell Spreading and Motility by Interacting with the Actin Cytoskeleton"

Article Title: Scapinin, the Protein Phosphatase 1 Binding Protein, Enhances Cell Spreading and Motility by Interacting with the Actin Cytoskeleton

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004247

The roles of the RPEL-repeat and PP1-binding domains in scapinin-cell spreading. ( A ) The pEGFP-scapinin mutants used in this study are illustrated. ( B ) Each construct shown in ( A ) was transfected into Cos7 cells. The cells were cultured for 24 hours and lysed in 0.5% Triton X-100/cytoskeleton buffer at 24 hours. GFP-scapinins were immunoprecipitated with anti-GFP antibody, and immunocomplexes were collected with protein A-agarose beads, washed, and subjected to Western blotting with anti-scapinin monoclonal antibody, anti-actin monoclonal antibody, and anti-PP1 polyclonal antibodies, respectively. ( C ) Each pEGFP-scapinin mutant was transfected into Cos7 cells. Cells were cultured for 16 hours and then monitored under a fluorescent microscope and photographed. Bar: 20 µm.
Figure Legend Snippet: The roles of the RPEL-repeat and PP1-binding domains in scapinin-cell spreading. ( A ) The pEGFP-scapinin mutants used in this study are illustrated. ( B ) Each construct shown in ( A ) was transfected into Cos7 cells. The cells were cultured for 24 hours and lysed in 0.5% Triton X-100/cytoskeleton buffer at 24 hours. GFP-scapinins were immunoprecipitated with anti-GFP antibody, and immunocomplexes were collected with protein A-agarose beads, washed, and subjected to Western blotting with anti-scapinin monoclonal antibody, anti-actin monoclonal antibody, and anti-PP1 polyclonal antibodies, respectively. ( C ) Each pEGFP-scapinin mutant was transfected into Cos7 cells. Cells were cultured for 16 hours and then monitored under a fluorescent microscope and photographed. Bar: 20 µm.

Techniques Used: Binding Assay, Construct, Transfection, Cell Culture, Immunoprecipitation, Western Blot, Mutagenesis, Microscopy

The RPEL repeats of scapinin interact with purified skeletal muscle actin and inhibit actin polymerization in vitro . ( A ) GST or GST-RPEL repeats (350–422 aa) were covalently conjugated to CNBr-agarose beads (as in Figure 1C ) and were incubated with purified skeletal muscle actin. After washing with RIPA buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mM Tris-HCl pH 8.0, 150 mM NaCl), bound proteins were eluted with SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and stained with coomassie brilliant blue (CBB). GST and GST-RPEL proteins (shown by a asterisk) were partly released from the beads by elution with SDS sample buffer. ( B ) Inhibition of actin polymerization by the RPEL repeats. Skeletal muscle actin (18 µM) was incubated with GST- RPEL repeats (350–422 aa) or GST at the indicated concentrations to polymerize at room temperature for 30 minutes, and then filamentous actin (P) and monomeric actin (S) were separated by ultracentrifugation. Aliquots were analyzed by SDS polyacrylamide gel electrophoresis and stained with coomassie brilliant blue (CBB). Since the contamination of supernatants in the pellet fraction was technically inevitable, a small portion of GST-RPEL construct was seen in the pellet. ( C ) The density of each actin band was measured by a densitometer and plotted.
Figure Legend Snippet: The RPEL repeats of scapinin interact with purified skeletal muscle actin and inhibit actin polymerization in vitro . ( A ) GST or GST-RPEL repeats (350–422 aa) were covalently conjugated to CNBr-agarose beads (as in Figure 1C ) and were incubated with purified skeletal muscle actin. After washing with RIPA buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mM Tris-HCl pH 8.0, 150 mM NaCl), bound proteins were eluted with SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and stained with coomassie brilliant blue (CBB). GST and GST-RPEL proteins (shown by a asterisk) were partly released from the beads by elution with SDS sample buffer. ( B ) Inhibition of actin polymerization by the RPEL repeats. Skeletal muscle actin (18 µM) was incubated with GST- RPEL repeats (350–422 aa) or GST at the indicated concentrations to polymerize at room temperature for 30 minutes, and then filamentous actin (P) and monomeric actin (S) were separated by ultracentrifugation. Aliquots were analyzed by SDS polyacrylamide gel electrophoresis and stained with coomassie brilliant blue (CBB). Since the contamination of supernatants in the pellet fraction was technically inevitable, a small portion of GST-RPEL construct was seen in the pellet. ( C ) The density of each actin band was measured by a densitometer and plotted.

Techniques Used: Purification, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Inhibition, Construct

Interaction of the RPEL repeats of scapinin with cellular actin. ( A ) The primary structure of scapinin. The N-terminal region that is conserved in the scapinin/phactr family (NC), the proline-rich region (PR), the three tandem repeats of PREL motifs (RPEL repeats 1, 2, and 3), and the PP1-binding domains (PP1) are indicated. In addition to the N-terminal conserved region (NC), the RPEL-repeat and PP1-binding domains are highly conserved in the scapinin/phactr family. ( B ) Three RPEL motifs are compared. The RPEL motif (pfam PF02755) is named according to the conserved amino acid sequence, RPxxxEL. ( C ) Interactions between the RPEL repeats of scapinin and cellular actin. The RPEL repeats of scapinin were produced as fusion proteins with glutathione S-transferase (GST) in E. Coli as illustrated. In the RP > AA mutant, both the arginine and proline residues in the second motif of the RPEL repeats were substituted with alanine residues. Purified GST-RPEL constructs were separated by SDS-polyacrylamide gel electrophoresis and stained with coomassie brilliant blue (CBB). GST-RPEL constructs were covalently conjugated to CNBr-agarose beads (5 mg protein/ml bed volume) and subjected to a pull-down assay with HL-60 cell lysates (see ‘ Methods ’). Actin in the pull-down-samples was determined by Western blotting (WB) with anti-actin antibody. The GST-RPEL construct (350–465 aa) contained substantial amounts of cleaved products (see ‘ Results ’).
Figure Legend Snippet: Interaction of the RPEL repeats of scapinin with cellular actin. ( A ) The primary structure of scapinin. The N-terminal region that is conserved in the scapinin/phactr family (NC), the proline-rich region (PR), the three tandem repeats of PREL motifs (RPEL repeats 1, 2, and 3), and the PP1-binding domains (PP1) are indicated. In addition to the N-terminal conserved region (NC), the RPEL-repeat and PP1-binding domains are highly conserved in the scapinin/phactr family. ( B ) Three RPEL motifs are compared. The RPEL motif (pfam PF02755) is named according to the conserved amino acid sequence, RPxxxEL. ( C ) Interactions between the RPEL repeats of scapinin and cellular actin. The RPEL repeats of scapinin were produced as fusion proteins with glutathione S-transferase (GST) in E. Coli as illustrated. In the RP > AA mutant, both the arginine and proline residues in the second motif of the RPEL repeats were substituted with alanine residues. Purified GST-RPEL constructs were separated by SDS-polyacrylamide gel electrophoresis and stained with coomassie brilliant blue (CBB). GST-RPEL constructs were covalently conjugated to CNBr-agarose beads (5 mg protein/ml bed volume) and subjected to a pull-down assay with HL-60 cell lysates (see ‘ Methods ’). Actin in the pull-down-samples was determined by Western blotting (WB) with anti-actin antibody. The GST-RPEL construct (350–465 aa) contained substantial amounts of cleaved products (see ‘ Results ’).

Techniques Used: Binding Assay, Sequencing, Produced, Mutagenesis, Purification, Construct, Polyacrylamide Gel Electrophoresis, Staining, Pull Down Assay, Western Blot

31) Product Images from "BRCA1 interacts with components of the histone deacetylase complex"

Article Title: BRCA1 interacts with components of the histone deacetylase complex

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

The BRCT domain binds to Rb-binding proteins in vitro . ( A ) Schematic representation of BRCA1 gene. The region used as a probe in the library screen is indicated. ( B ) GST pull-down assays demonstrating that BRCT domain interacts with partial polypeptide of RbAp46 and full-length RbAp46 and RbAp48 proteins. In vitro -translated, 35 S-labeled BRCT (10 μl) was incubated with 20 μl of glutathione-Sepharose beads and an equal amount of GST or GST-fusion proteins, as indicated. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE, and visualized by using autoradiography. A portion of the in vitro -translated, 35 S-labeled BRCT, corresponding to ≈20% of the labeled protein in the binding reaction, was loaded as “Input”. ( C ) Schematic representation of the BRCT repeats. Mutations analyzed for in vitro binding are indicated. ( D ) Mutations in BRCT domain interfere with interaction between RbAp46 and BRCT. GST pull-down assays were performed as in A with the wild type or mutation-containing BRCT polypeptides as indicated. Approximately 5% of each in vitro -translated protein in the binding reactions were loaded as Input.
Figure Legend Snippet: The BRCT domain binds to Rb-binding proteins in vitro . ( A ) Schematic representation of BRCA1 gene. The region used as a probe in the library screen is indicated. ( B ) GST pull-down assays demonstrating that BRCT domain interacts with partial polypeptide of RbAp46 and full-length RbAp46 and RbAp48 proteins. In vitro -translated, 35 S-labeled BRCT (10 μl) was incubated with 20 μl of glutathione-Sepharose beads and an equal amount of GST or GST-fusion proteins, as indicated. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE, and visualized by using autoradiography. A portion of the in vitro -translated, 35 S-labeled BRCT, corresponding to ≈20% of the labeled protein in the binding reaction, was loaded as “Input”. ( C ) Schematic representation of the BRCT repeats. Mutations analyzed for in vitro binding are indicated. ( D ) Mutations in BRCT domain interfere with interaction between RbAp46 and BRCT. GST pull-down assays were performed as in A with the wild type or mutation-containing BRCT polypeptides as indicated. Approximately 5% of each in vitro -translated protein in the binding reactions were loaded as Input.

Techniques Used: Binding Assay, In Vitro, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography, Mutagenesis

Rb interacts with the BRCT domain. ( A ) GST pull-down experiments show that Rb interacts with BRCT domain in the presence or absence of partial RbAp46 polypeptide. In vitro -translated, 35 S-labeled BRCT (12.5 μl) and partial RbAp46 polypeptides were incubated individually or in combination with 20 μl of glutathione-Sepharose beads with equal amount of GST-Rb or GST-Rb pocket mutation fusion proteins as indicated. GST-coated beads were incubated with each polypeptide separately. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE and visualized by using autoradiography. Approximately 20% of labeled protein in the binding reaction were loaded as Input. ( B ) Colocalization of BRCA1 and Rb. HeLa cells were prepared as described in Material and Methods , stained with a mouse mAb against Rb, IF8, (green in a ), a rabbit polyclonal antibody against BRCA1, I-20, (red in b ). The regions of overlap between red and green signals appear as yellow ( c ), indicating colocalization of BRCA1 and Rb. The nucleus of each cells are shown by DAPI staining ( d ).
Figure Legend Snippet: Rb interacts with the BRCT domain. ( A ) GST pull-down experiments show that Rb interacts with BRCT domain in the presence or absence of partial RbAp46 polypeptide. In vitro -translated, 35 S-labeled BRCT (12.5 μl) and partial RbAp46 polypeptides were incubated individually or in combination with 20 μl of glutathione-Sepharose beads with equal amount of GST-Rb or GST-Rb pocket mutation fusion proteins as indicated. GST-coated beads were incubated with each polypeptide separately. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE and visualized by using autoradiography. Approximately 20% of labeled protein in the binding reaction were loaded as Input. ( B ) Colocalization of BRCA1 and Rb. HeLa cells were prepared as described in Material and Methods , stained with a mouse mAb against Rb, IF8, (green in a ), a rabbit polyclonal antibody against BRCA1, I-20, (red in b ). The regions of overlap between red and green signals appear as yellow ( c ), indicating colocalization of BRCA1 and Rb. The nucleus of each cells are shown by DAPI staining ( d ).

Techniques Used: In Vitro, Labeling, Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis, Autoradiography, Binding Assay, Staining

The BRCT domain associates with HDAC1 and HDAC2. Western blot analysis of GST pull-down assays using antibodies against HDAC1 and HDAC2. HeLa whole-cell lysates (300 μg) were incubated with 20 μl of glutathione-Sepharose beads covered with equal amounts of GST, GST-BRCT (amino acids 1,536–1,863), or GST-NH 2 -BRCA1 (amino acids 1–304) fusion proteins. Bound proteins were resolved on SDS/10% PAGE, transferred to nitrocellulose membranes, and blotted with HDAC1 or HDAC2 Abs.
Figure Legend Snippet: The BRCT domain associates with HDAC1 and HDAC2. Western blot analysis of GST pull-down assays using antibodies against HDAC1 and HDAC2. HeLa whole-cell lysates (300 μg) were incubated with 20 μl of glutathione-Sepharose beads covered with equal amounts of GST, GST-BRCT (amino acids 1,536–1,863), or GST-NH 2 -BRCA1 (amino acids 1–304) fusion proteins. Bound proteins were resolved on SDS/10% PAGE, transferred to nitrocellulose membranes, and blotted with HDAC1 or HDAC2 Abs.

Techniques Used: Western Blot, Incubation, Polyacrylamide Gel Electrophoresis

32) Product Images from "The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity"

Article Title: The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2008-0098OC

( A ) Circular dichroism of RTS-generated PCRan1p conjugated to V5 beads was used to determine a thermal denaturation and refolding profile by measuring ellipticity at 222 nm from 4 to 45°C. In addition, V5 beads were assayed alone to determine if noise was produced as a result of interference of the beads in the solution. Diamonds , V5 beads; squares , PCRan1 unfolding; triangles , PCRan1 refolding. ( B ) Far-ultraviolet (UV) spectra of RTS-generated PCRan1p conjugated to V5 beads were determined at five different temperatures ranging from 10 to 45°C, and the minima were plotted to demonstrate the trend line . ( C ) Fluorescence spectroscopy, with an excitation at 294 nm and emission measured from 310 to 400 nm, was performed to determine the conformational profile of RTS-generated PCRan1p conjugated to V5 beads. A V5 bead control was subtracted from all samples and then plotted at their respective maxima. ( D ) RTS-generated PCRan1p conjugated to V5 agarose beads was incubated at 25, 30, 37, or 45°C for 1 hour, followed by performing an in vitro kinase assay at 25 or 45°C for 1 hour to determine the reversibility of the unfolding reaction using PHAS-I as the substrate.
Figure Legend Snippet: ( A ) Circular dichroism of RTS-generated PCRan1p conjugated to V5 beads was used to determine a thermal denaturation and refolding profile by measuring ellipticity at 222 nm from 4 to 45°C. In addition, V5 beads were assayed alone to determine if noise was produced as a result of interference of the beads in the solution. Diamonds , V5 beads; squares , PCRan1 unfolding; triangles , PCRan1 refolding. ( B ) Far-ultraviolet (UV) spectra of RTS-generated PCRan1p conjugated to V5 beads were determined at five different temperatures ranging from 10 to 45°C, and the minima were plotted to demonstrate the trend line . ( C ) Fluorescence spectroscopy, with an excitation at 294 nm and emission measured from 310 to 400 nm, was performed to determine the conformational profile of RTS-generated PCRan1p conjugated to V5 beads. A V5 bead control was subtracted from all samples and then plotted at their respective maxima. ( D ) RTS-generated PCRan1p conjugated to V5 agarose beads was incubated at 25, 30, 37, or 45°C for 1 hour, followed by performing an in vitro kinase assay at 25 or 45°C for 1 hour to determine the reversibility of the unfolding reaction using PHAS-I as the substrate.

Techniques Used: Generated, Produced, Fluorescence, Spectroscopy, Incubation, In Vitro, Kinase Assay

( A ) PCRan1p immunoprecipitated from Pneumocystis carinii cysts and trophic forms purified from infected rats, using a specific PCRan1p antibody and protein A sepharose, was then added to a kinase assay performed at a series of temperatures to determine enzymatic activity of in vivo –derived PCRan1p. Western blots of PCRan1p using a specific antibody were performed as a loading control. ( B ) P. carinii isolated from infected rats was incubated for 2 hours at a series of temperatures, followed by protein isolation and Western blotting using a specific PCRan1p antibody. PCCBK1 was used as a loading control to verify equal protein loading.
Figure Legend Snippet: ( A ) PCRan1p immunoprecipitated from Pneumocystis carinii cysts and trophic forms purified from infected rats, using a specific PCRan1p antibody and protein A sepharose, was then added to a kinase assay performed at a series of temperatures to determine enzymatic activity of in vivo –derived PCRan1p. Western blots of PCRan1p using a specific antibody were performed as a loading control. ( B ) P. carinii isolated from infected rats was incubated for 2 hours at a series of temperatures, followed by protein isolation and Western blotting using a specific PCRan1p antibody. PCCBK1 was used as a loading control to verify equal protein loading.

Techniques Used: Immunoprecipitation, Purification, Infection, Kinase Assay, Activity Assay, In Vivo, Derivative Assay, Western Blot, Isolation, Incubation

( A ) V5-tagged agarose beads used to immunoprecipitate PCRan1p or Pat1p, followed by Western blotting using the V5 antibody on Rapid Translation System (RTS)-generated protein made from pET102-TOPO plus PCRan1 or Pat1 . ( B ) In vitro kinase assay on RTS-generated PCRan1p and Pat1p performed at 25°C for 1 hour using PHAS-I as a substrate. ( C ) In vitro kinase assay using RTS-generated PCRan1p and Pat1p assayed for enzymatic activity at a variety of different temperatures. V5 antibody was used to Western blot for overall protein levels of either PCRan1p or Pat1p.
Figure Legend Snippet: ( A ) V5-tagged agarose beads used to immunoprecipitate PCRan1p or Pat1p, followed by Western blotting using the V5 antibody on Rapid Translation System (RTS)-generated protein made from pET102-TOPO plus PCRan1 or Pat1 . ( B ) In vitro kinase assay on RTS-generated PCRan1p and Pat1p performed at 25°C for 1 hour using PHAS-I as a substrate. ( C ) In vitro kinase assay using RTS-generated PCRan1p and Pat1p assayed for enzymatic activity at a variety of different temperatures. V5 antibody was used to Western blot for overall protein levels of either PCRan1p or Pat1p.

Techniques Used: Western Blot, Generated, In Vitro, Kinase Assay, Activity Assay

33) Product Images from "SECIS-binding protein 2 interacts with the SMN complex and the methylosome for selenoprotein mRNP assembly and translation"

Article Title: SECIS-binding protein 2 interacts with the SMN complex and the methylosome for selenoprotein mRNP assembly and translation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx031

Reconstitution and activity of the SBP2/methylosome complex and functional analysis ( A ) Coexpression and purification of SBP2 associated with the methylosome complex. Recombinant baculoviruses allowing the expression of GST-SBP2, PRMT5, MEP50 and HA-pICln were used to infect Sf9 insect cells in the combinations indicated. Expression of PRMT5/MEP50 and HA-pICln alone served as negative controls. The complexes associated to GST-SBP2 were purified on Glutathione Sepharose, and the associated proteins were analyzed by SDS-PAGE and western blotting using the indicated antibodies. In: input (4% of the total cell extract). ( B ) SBP2 is not a substrate of PRMT5/MEP50. For methylation assays PRMT5/MEP50 was incubated with SBP2 or histones (H3-H4) 2 in the presence of 14 C SAM for 30 min at 37°C. The (H3-H4) 2 tetramer is a methylation substrate of the methylosome and therefore serves as a positive control. Proteins are analyzed on SDS-PAGE and revealed by coomassie staining (lanes 1–3). The radioactive methylation signal is revealed by phosphorImager after overnight exposure (lanes 4–6). The cartoon represents His-Tag SBP2 and the position of the RG sequences (*) that are potential sites of methylation. L: ladder. ( C ) Sm proteins do not bind selenoprotein mRNAs. Total RNA extracted from HEK293FT cells was immunoprecipitated with anti-Sm antibodies. qRT-PCR was used to determine the RNA enrichment in the IP experiment compared to the input extract by the ΔΔCt method. SelR, GPx4, SelM, SelT, TrxR1 and Sel15 are selenoprotein mRNAs. U1 and U2 snRNAs were used as positive controls; ß-actin and LDHA mRNAs are housekeeping mRNAs used as negative controls. Error bars represent standard deviation of an average of three independent experiments.
Figure Legend Snippet: Reconstitution and activity of the SBP2/methylosome complex and functional analysis ( A ) Coexpression and purification of SBP2 associated with the methylosome complex. Recombinant baculoviruses allowing the expression of GST-SBP2, PRMT5, MEP50 and HA-pICln were used to infect Sf9 insect cells in the combinations indicated. Expression of PRMT5/MEP50 and HA-pICln alone served as negative controls. The complexes associated to GST-SBP2 were purified on Glutathione Sepharose, and the associated proteins were analyzed by SDS-PAGE and western blotting using the indicated antibodies. In: input (4% of the total cell extract). ( B ) SBP2 is not a substrate of PRMT5/MEP50. For methylation assays PRMT5/MEP50 was incubated with SBP2 or histones (H3-H4) 2 in the presence of 14 C SAM for 30 min at 37°C. The (H3-H4) 2 tetramer is a methylation substrate of the methylosome and therefore serves as a positive control. Proteins are analyzed on SDS-PAGE and revealed by coomassie staining (lanes 1–3). The radioactive methylation signal is revealed by phosphorImager after overnight exposure (lanes 4–6). The cartoon represents His-Tag SBP2 and the position of the RG sequences (*) that are potential sites of methylation. L: ladder. ( C ) Sm proteins do not bind selenoprotein mRNAs. Total RNA extracted from HEK293FT cells was immunoprecipitated with anti-Sm antibodies. qRT-PCR was used to determine the RNA enrichment in the IP experiment compared to the input extract by the ΔΔCt method. SelR, GPx4, SelM, SelT, TrxR1 and Sel15 are selenoprotein mRNAs. U1 and U2 snRNAs were used as positive controls; ß-actin and LDHA mRNAs are housekeeping mRNAs used as negative controls. Error bars represent standard deviation of an average of three independent experiments.

Techniques Used: Activity Assay, Functional Assay, Purification, Recombinant, Expressing, SDS Page, Western Blot, Methylation, Incubation, Positive Control, Staining, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation

The methylosome complex interacts with SBP2 in vivo . ( A ) Immunopurification of endogenous SBP2 from HeLa cytoplasmic extracts using antipeptide antibodies (α-pepSBP2) directed against SBP2 residues 380–852. PI: beads with preimmune serum. The immunopurified proteins were identified by mass spectrometry (MS) and specific proteins are indicated on the right. Numbers represent common sepharose-matrix binding contaminants found in MS analysis ( 56 ), most of them are cytoskeletal proteins (1,1΄: Dynein chains, 2: Keratin, 3: Tubulin ß; 4: Tubulin α; 5: VASP actin associated protein). Molecular weight (kDa) is indicated. ( B–D ) Co-immunoprecipitations using anti-GFP beads and HEK293FT cells transfected by (B and C) GFP-SBP2 and (D) GFP-PRMT5, GFP-MEP50, GFP-pICln or GFP alone. Immunoprecipitations were performed in high salt (300 mM NaCl) and low salt (150 mM NaCl) conditions. The immunoprecipitated endogenous proteins were analyzed by SDS-PAGE and western blotting using the indicated antibodies. In: input (10% of total). * smear of unknown origin always present at high salt but absent at low salt. ( E ) Direct interactions between SBP2 and components of the methylosome complex. Y2H interaction tests performed in Saccharomyces cerevisiae AH109 between SBP2 and the methylosome components PRMT5, MEP50 and pICln; Nufip was used as a positive interaction control with SBP2. Experiments were performed as described Figure 1B . Data are shown in Supplementary Figure S1A .
Figure Legend Snippet: The methylosome complex interacts with SBP2 in vivo . ( A ) Immunopurification of endogenous SBP2 from HeLa cytoplasmic extracts using antipeptide antibodies (α-pepSBP2) directed against SBP2 residues 380–852. PI: beads with preimmune serum. The immunopurified proteins were identified by mass spectrometry (MS) and specific proteins are indicated on the right. Numbers represent common sepharose-matrix binding contaminants found in MS analysis ( 56 ), most of them are cytoskeletal proteins (1,1΄: Dynein chains, 2: Keratin, 3: Tubulin ß; 4: Tubulin α; 5: VASP actin associated protein). Molecular weight (kDa) is indicated. ( B–D ) Co-immunoprecipitations using anti-GFP beads and HEK293FT cells transfected by (B and C) GFP-SBP2 and (D) GFP-PRMT5, GFP-MEP50, GFP-pICln or GFP alone. Immunoprecipitations were performed in high salt (300 mM NaCl) and low salt (150 mM NaCl) conditions. The immunoprecipitated endogenous proteins were analyzed by SDS-PAGE and western blotting using the indicated antibodies. In: input (10% of total). * smear of unknown origin always present at high salt but absent at low salt. ( E ) Direct interactions between SBP2 and components of the methylosome complex. Y2H interaction tests performed in Saccharomyces cerevisiae AH109 between SBP2 and the methylosome components PRMT5, MEP50 and pICln; Nufip was used as a positive interaction control with SBP2. Experiments were performed as described Figure 1B . Data are shown in Supplementary Figure S1A .

Techniques Used: In Vivo, Immu-Puri, Mass Spectrometry, Binding Assay, Molecular Weight, Transfection, Immunoprecipitation, SDS Page, Western Blot

34) Product Images from "Combination of a Zinc Finger and Homeodomain Required for Protein-Interaction"

Article Title: Combination of a Zinc Finger and Homeodomain Required for Protein-Interaction

Journal: Molecular biology reports

doi:

The Zfhep HD alone does not bind GST-POU or GST-POU. 32 HD P-HD (5 × 10 5 cpm) was incubated with 20 μl of either GST, GST-HD, GST-POU HD or GST-POU bound to glutathione sepharose, and washed as described. 32 P-HD bound by the fusion protein was eluted in SDS sample buffer, separated by 15 % SDS-PAGE and analyzed by autoradiography. Five percent of the 32 P-HD input was loaded in lane 1 for comparison. Zfhep HD did not form homodimers under these conditions, as indicated by a lack of binding to GST-HD, nor did 32 P-HD bind to Oct-1 proteins.
Figure Legend Snippet: The Zfhep HD alone does not bind GST-POU or GST-POU. 32 HD P-HD (5 × 10 5 cpm) was incubated with 20 μl of either GST, GST-HD, GST-POU HD or GST-POU bound to glutathione sepharose, and washed as described. 32 P-HD bound by the fusion protein was eluted in SDS sample buffer, separated by 15 % SDS-PAGE and analyzed by autoradiography. Five percent of the 32 P-HD input was loaded in lane 1 for comparison. Zfhep HD did not form homodimers under these conditions, as indicated by a lack of binding to GST-HD, nor did 32 P-HD bind to Oct-1 proteins.

Techniques Used: Incubation, SDS Page, Autoradiography, Binding Assay

Zfhep ZHD binds the Oct-1 POU domain. 32 P-ZHD (5 × 10 5 cpm) was incubated with 20 μl GST, GST-POU HD or GST-POU bound to glutathione-sepharose in the presence of 4 mg E. coli extract as competitor. Pellets were washed as described. 32 P-ZHD bound by the fusion protein was eluted in SDS sample buffer, separated by 12.5 % SDS-PAGE and analyzed by autoradiography. Lane 1 received 5 % 32 P-ZHD input in the reaction (Input). Full-length 32 P-ZHD is indicated by the arrow.
Figure Legend Snippet: Zfhep ZHD binds the Oct-1 POU domain. 32 P-ZHD (5 × 10 5 cpm) was incubated with 20 μl GST, GST-POU HD or GST-POU bound to glutathione-sepharose in the presence of 4 mg E. coli extract as competitor. Pellets were washed as described. 32 P-ZHD bound by the fusion protein was eluted in SDS sample buffer, separated by 12.5 % SDS-PAGE and analyzed by autoradiography. Lane 1 received 5 % 32 P-ZHD input in the reaction (Input). Full-length 32 P-ZHD is indicated by the arrow.

Techniques Used: Incubation, SDS Page, Autoradiography

ZHD interaction with Oct-1 POU domain is specific. 32 P-ZHD (2.5 μl, approximately 2.3 × 10 5 cpm) was incubated with 7 μl glutathione-sepharose beads with GST-POU (or GST alone) with or without a 100-fold excess non-radioactive ZHD (250 μl). Beads were washed and eluted as described. The SDS-PAGE gel lanes received either 5 % 32 P-ZHD input (Input), or 32 P-ZHD eluted from the GST beads (GST), GST-POU beads (POU), or GST-POU beads with 100-fold excess non-radioactive ZHD (100x).
Figure Legend Snippet: ZHD interaction with Oct-1 POU domain is specific. 32 P-ZHD (2.5 μl, approximately 2.3 × 10 5 cpm) was incubated with 7 μl glutathione-sepharose beads with GST-POU (or GST alone) with or without a 100-fold excess non-radioactive ZHD (250 μl). Beads were washed and eluted as described. The SDS-PAGE gel lanes received either 5 % 32 P-ZHD input (Input), or 32 P-ZHD eluted from the GST beads (GST), GST-POU beads (POU), or GST-POU beads with 100-fold excess non-radioactive ZHD (100x).

Techniques Used: Incubation, SDS Page

35) Product Images from "The Chloroplast SRP Systems of Chaetosphaeridium globosum and Physcomitrella patens as Intermediates in the Evolution of SRP-Dependent Protein Transport in Higher Plants"

Article Title: The Chloroplast SRP Systems of Chaetosphaeridium globosum and Physcomitrella patens as Intermediates in the Evolution of SRP-Dependent Protein Transport in Higher Plants

Journal: PLoS ONE

doi: 10.1371/journal.pone.0166818

Interaction analysis between cpSRP54M and various cpSRP43 constructs of Chaetosphaeridium globosum . (A) In vitro pull-down assays were performed as described previously [ 15 ]. Combinations of recombinant GST-cpSRP43 (At-, Cg-GST-43) or the mutant construct Cg-GST-43(V192P) and His-tagged cpSRP54M (At-, Cg-His-54M) proteins were analyzed as indicated using glutathione-sepharose. Control reactions were performed with recombinant GST. One-tenth of the loaded proteins (upper panel) and one-third of eluted proteins (lower panel) were analyzed by SDS-PAGE and Coomassie staining. The asterisk (*) indicates the used His-tagged constructs. (B) Protein-protein interactions between His-tagged Chaetosphaeridium globosum cpSRP54M (Cg-54M) and cpSRP43 (Cg-43) or cpSRP43(V192P) were analyzed by size exclusion chromatography using equimolar amounts of the indicated recombinant proteins: (green) Cg-His-43 and Cg-His-54M, (orange) Cg-His-43(V192P) and Cg-His-54M, (blue) Cg-His-43, (violet) Cg-His-43(V192P), and (red) Cg-His-54M. Elution fractions in a range from 8.5 to 14.5 ml were separated by SDS-PAGE and detected by Coomassie staining.
Figure Legend Snippet: Interaction analysis between cpSRP54M and various cpSRP43 constructs of Chaetosphaeridium globosum . (A) In vitro pull-down assays were performed as described previously [ 15 ]. Combinations of recombinant GST-cpSRP43 (At-, Cg-GST-43) or the mutant construct Cg-GST-43(V192P) and His-tagged cpSRP54M (At-, Cg-His-54M) proteins were analyzed as indicated using glutathione-sepharose. Control reactions were performed with recombinant GST. One-tenth of the loaded proteins (upper panel) and one-third of eluted proteins (lower panel) were analyzed by SDS-PAGE and Coomassie staining. The asterisk (*) indicates the used His-tagged constructs. (B) Protein-protein interactions between His-tagged Chaetosphaeridium globosum cpSRP54M (Cg-54M) and cpSRP43 (Cg-43) or cpSRP43(V192P) were analyzed by size exclusion chromatography using equimolar amounts of the indicated recombinant proteins: (green) Cg-His-43 and Cg-His-54M, (orange) Cg-His-43(V192P) and Cg-His-54M, (blue) Cg-His-43, (violet) Cg-His-43(V192P), and (red) Cg-His-54M. Elution fractions in a range from 8.5 to 14.5 ml were separated by SDS-PAGE and detected by Coomassie staining.

Techniques Used: Construct, In Vitro, Recombinant, Mutagenesis, SDS Page, Staining, Size-exclusion Chromatography

Interaction analysis between cpSRP54 and various cpSRP43 constructs of Arabidopsis thaliana and Physcomitrella patens . (A) Yeast two-hybrid interaction studies. For yeast two-hybrid assays, the yeast strain Y190 was co-transformed with pGBKT7 constructs encoding full-length cpSRP54 (54) and pACT2 constructs encoding cpSRP43 (43) or the indicated cpSRP43 mutants of Arabidopsis thaliana (At) and Physcomitrella patens (Pp). Co-transformed cells were dotted onto minimal media lacking Leu and Trp (-LT) to check for co-transformation, or lacking Leu, Trp and His (-LTH) to assess interaction. Negative controls were conducted with an empty vector (pGBKT7 or pACT2). (B) In vitro pull-down assays were performed with recombinant GST-cpSRP43 (At-, Pp-GST-43) constructs and His-tagged cpSRP54 proteins (At-His-54, Pp-54-His) as indicated, using glutathione-sepharose. Control reactions were performed with recombinant GST. One-tenth of the loaded proteins (upper panel) and one-third of eluted proteins (lower panel) were separated using SDS-PAGE and detected using Coomassie staining. The asterisk (*) indicates the used His-tagged constructs.
Figure Legend Snippet: Interaction analysis between cpSRP54 and various cpSRP43 constructs of Arabidopsis thaliana and Physcomitrella patens . (A) Yeast two-hybrid interaction studies. For yeast two-hybrid assays, the yeast strain Y190 was co-transformed with pGBKT7 constructs encoding full-length cpSRP54 (54) and pACT2 constructs encoding cpSRP43 (43) or the indicated cpSRP43 mutants of Arabidopsis thaliana (At) and Physcomitrella patens (Pp). Co-transformed cells were dotted onto minimal media lacking Leu and Trp (-LT) to check for co-transformation, or lacking Leu, Trp and His (-LTH) to assess interaction. Negative controls were conducted with an empty vector (pGBKT7 or pACT2). (B) In vitro pull-down assays were performed with recombinant GST-cpSRP43 (At-, Pp-GST-43) constructs and His-tagged cpSRP54 proteins (At-His-54, Pp-54-His) as indicated, using glutathione-sepharose. Control reactions were performed with recombinant GST. One-tenth of the loaded proteins (upper panel) and one-third of eluted proteins (lower panel) were separated using SDS-PAGE and detected using Coomassie staining. The asterisk (*) indicates the used His-tagged constructs.

Techniques Used: Construct, Transformation Assay, Plasmid Preparation, In Vitro, Recombinant, SDS Page, Staining

36) Product Images from "TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production"

Article Title: TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq264

Rrn3p levels are reduced upon TOR inactivation and proteasome-dependent degradation. ( A ) Rrn3p is degraded upon TOR inactivation and inhibition of translation. Yeast strain RRN3-Prot.A expressing a chromosomally Prot.A-tagged Rrn3p was grown in YPD at 30°C to mid-log phase (OD 600 ≈ 0.4; t = 0 min), before the cells were either treated with 200 ng/ml of rapamycin or with 100 µg/ml of cycloheximide, or starved in SDC-Leu (aa-depletion), respectively. At the time points indicated cells were collected and lysed. Same amounts of WCE (20 µg) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p and the Pol I specific subunit A43, respectively. ( B ) Rrn3p-degradation depends on proteasome activity. The proteasome ts-mutant strain ( cim3-1, TOY 652 ) expressing a chromosomally TAP-tagged Rrn3p or the isogenic CIM3 WT strain (WT, TOY 651 ) were grown to mid-log phase in YPD at 24°C ( t = 0 min), before the cells were starved at 37°C in SDC-Leu medium (-Leu). At the time points indicated cells were collected and lysed. Same amounts of WCE (30 µg) were analysed by western blotting using antibodies directed against the TAP-tag of Rrn3p and the Pol I subunit A135, respectively. ( C ) Rrn3p is ubiquitylated. Yeast strain pNOP1-RRN3-Prot.A, expressing Prot.A-tagged Rrn3p from a plasmid was grown in YPD at 30°C to midlog phase, before half of the cells were treated with 200 ng/ml of rapamycin for 10 min. Cells of rapamycin treated and untreated cultures were collected and lysed. Same amounts of WCEs (6 mg) were incubated with either recombinant GST-Dsk2p, or recombinant GST immobilized on 50 µl of glutathione sepharose. After washing, proteins bound to the beads were eluted with SDS sample buffer. Same amounts (1%) (50 µg) of input (IN) and flow through (FT), 0.5% of the wash steps (washes) and 50% of the eluate (E) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p.
Figure Legend Snippet: Rrn3p levels are reduced upon TOR inactivation and proteasome-dependent degradation. ( A ) Rrn3p is degraded upon TOR inactivation and inhibition of translation. Yeast strain RRN3-Prot.A expressing a chromosomally Prot.A-tagged Rrn3p was grown in YPD at 30°C to mid-log phase (OD 600 ≈ 0.4; t = 0 min), before the cells were either treated with 200 ng/ml of rapamycin or with 100 µg/ml of cycloheximide, or starved in SDC-Leu (aa-depletion), respectively. At the time points indicated cells were collected and lysed. Same amounts of WCE (20 µg) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p and the Pol I specific subunit A43, respectively. ( B ) Rrn3p-degradation depends on proteasome activity. The proteasome ts-mutant strain ( cim3-1, TOY 652 ) expressing a chromosomally TAP-tagged Rrn3p or the isogenic CIM3 WT strain (WT, TOY 651 ) were grown to mid-log phase in YPD at 24°C ( t = 0 min), before the cells were starved at 37°C in SDC-Leu medium (-Leu). At the time points indicated cells were collected and lysed. Same amounts of WCE (30 µg) were analysed by western blotting using antibodies directed against the TAP-tag of Rrn3p and the Pol I subunit A135, respectively. ( C ) Rrn3p is ubiquitylated. Yeast strain pNOP1-RRN3-Prot.A, expressing Prot.A-tagged Rrn3p from a plasmid was grown in YPD at 30°C to midlog phase, before half of the cells were treated with 200 ng/ml of rapamycin for 10 min. Cells of rapamycin treated and untreated cultures were collected and lysed. Same amounts of WCEs (6 mg) were incubated with either recombinant GST-Dsk2p, or recombinant GST immobilized on 50 µl of glutathione sepharose. After washing, proteins bound to the beads were eluted with SDS sample buffer. Same amounts (1%) (50 µg) of input (IN) and flow through (FT), 0.5% of the wash steps (washes) and 50% of the eluate (E) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p.

Techniques Used: Inhibition, Expressing, Western Blot, Activity Assay, Mutagenesis, Plasmid Preparation, Incubation, Recombinant, Flow Cytometry

Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. ( A ) Gelfiltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were grown in YPD at 30°C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 µg) were separated on a Superose-6® column in a buffer containing 1.5 M potassium acetate. An amount of 250 µl of the collected 500 µl fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 µg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel filtration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. ( B ) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 (ΔN), both expressing chromosomally HA 3 -tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed ( t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above ( t = 120 min). The HA 3 -tagged Pol I subunit A43 was immunoprecipitated (α-HA-IPs) from 250 µl of extracts (Inputs) with anti-HA antibody. Fifty percent of the α-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3-ΔN-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantified using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. ( C ) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN), both expressing either chromosomally HA 3 -tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and sonified. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA 3 -tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of specific DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers specific for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. ( D ) Reduction of 35S pre-rRNA synthesis is attenuated in the ΔN-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were cultured to mid-log phase ( t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 µCi of [ 5 , 6- 3 H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3 H-labelled RNAs were visualized and quantified using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32 P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantified as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the different rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100.
Figure Legend Snippet: Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. ( A ) Gelfiltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were grown in YPD at 30°C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 µg) were separated on a Superose-6® column in a buffer containing 1.5 M potassium acetate. An amount of 250 µl of the collected 500 µl fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 µg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel filtration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. ( B ) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 (ΔN), both expressing chromosomally HA 3 -tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed ( t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above ( t = 120 min). The HA 3 -tagged Pol I subunit A43 was immunoprecipitated (α-HA-IPs) from 250 µl of extracts (Inputs) with anti-HA antibody. Fifty percent of the α-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3-ΔN-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantified using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. ( C ) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN), both expressing either chromosomally HA 3 -tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and sonified. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA 3 -tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of specific DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers specific for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. ( D ) Reduction of 35S pre-rRNA synthesis is attenuated in the ΔN-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were cultured to mid-log phase ( t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 µCi of [ 5 , 6- 3 H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3 H-labelled RNAs were visualized and quantified using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32 P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantified as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the different rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100.

Techniques Used: Cell Culture, Centrifugation, Lysis, Western Blot, Filtration, Expressing, Immunoprecipitation, Imaging, Software, Chromatin Immunoprecipitation, DNA Extraction, Real-time Polymerase Chain Reaction, Mutagenesis, Isolation, Agarose Gel Electrophoresis, Northern Blot

37) Product Images from "TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production"

Article Title: TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq264

Rrn3p levels are reduced upon TOR inactivation and proteasome-dependent degradation. ( A ) Rrn3p is degraded upon TOR inactivation and inhibition of translation. Yeast strain RRN3-Prot.A expressing a chromosomally Prot.A-tagged Rrn3p was grown in YPD at 30°C to mid-log phase (OD 600 ≈ 0.4; t = 0 min), before the cells were either treated with 200 ng/ml of rapamycin or with 100 µg/ml of cycloheximide, or starved in SDC-Leu (aa-depletion), respectively. At the time points indicated cells were collected and lysed. Same amounts of WCE (20 µg) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p and the Pol I specific subunit A43, respectively. ( B ) Rrn3p-degradation depends on proteasome activity. The proteasome ts-mutant strain ( cim3-1, TOY 652 ) expressing a chromosomally TAP-tagged Rrn3p or the isogenic CIM3 WT strain (WT, TOY 651 ) were grown to mid-log phase in YPD at 24°C ( t = 0 min), before the cells were starved at 37°C in SDC-Leu medium (-Leu). At the time points indicated cells were collected and lysed. Same amounts of WCE (30 µg) were analysed by western blotting using antibodies directed against the TAP-tag of Rrn3p and the Pol I subunit A135, respectively. ( C ) Rrn3p is ubiquitylated. Yeast strain pNOP1-RRN3-Prot.A, expressing Prot.A-tagged Rrn3p from a plasmid was grown in YPD at 30°C to midlog phase, before half of the cells were treated with 200 ng/ml of rapamycin for 10 min. Cells of rapamycin treated and untreated cultures were collected and lysed. Same amounts of WCEs (6 mg) were incubated with either recombinant GST-Dsk2p, or recombinant GST immobilized on 50 µl of glutathione sepharose. After washing, proteins bound to the beads were eluted with SDS sample buffer. Same amounts (1%) (50 µg) of input (IN) and flow through (FT), 0.5% of the wash steps (washes) and 50% of the eluate (E) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p.
Figure Legend Snippet: Rrn3p levels are reduced upon TOR inactivation and proteasome-dependent degradation. ( A ) Rrn3p is degraded upon TOR inactivation and inhibition of translation. Yeast strain RRN3-Prot.A expressing a chromosomally Prot.A-tagged Rrn3p was grown in YPD at 30°C to mid-log phase (OD 600 ≈ 0.4; t = 0 min), before the cells were either treated with 200 ng/ml of rapamycin or with 100 µg/ml of cycloheximide, or starved in SDC-Leu (aa-depletion), respectively. At the time points indicated cells were collected and lysed. Same amounts of WCE (20 µg) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p and the Pol I specific subunit A43, respectively. ( B ) Rrn3p-degradation depends on proteasome activity. The proteasome ts-mutant strain ( cim3-1, TOY 652 ) expressing a chromosomally TAP-tagged Rrn3p or the isogenic CIM3 WT strain (WT, TOY 651 ) were grown to mid-log phase in YPD at 24°C ( t = 0 min), before the cells were starved at 37°C in SDC-Leu medium (-Leu). At the time points indicated cells were collected and lysed. Same amounts of WCE (30 µg) were analysed by western blotting using antibodies directed against the TAP-tag of Rrn3p and the Pol I subunit A135, respectively. ( C ) Rrn3p is ubiquitylated. Yeast strain pNOP1-RRN3-Prot.A, expressing Prot.A-tagged Rrn3p from a plasmid was grown in YPD at 30°C to midlog phase, before half of the cells were treated with 200 ng/ml of rapamycin for 10 min. Cells of rapamycin treated and untreated cultures were collected and lysed. Same amounts of WCEs (6 mg) were incubated with either recombinant GST-Dsk2p, or recombinant GST immobilized on 50 µl of glutathione sepharose. After washing, proteins bound to the beads were eluted with SDS sample buffer. Same amounts (1%) (50 µg) of input (IN) and flow through (FT), 0.5% of the wash steps (washes) and 50% of the eluate (E) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p.

Techniques Used: Inhibition, Expressing, Western Blot, Activity Assay, Mutagenesis, Plasmid Preparation, Incubation, Recombinant, Flow Cytometry

Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. ( A ) Gelfiltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were grown in YPD at 30°C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 µg) were separated on a Superose-6® column in a buffer containing 1.5 M potassium acetate. An amount of 250 µl of the collected 500 µl fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 µg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel filtration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. ( B ) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 (ΔN), both expressing chromosomally HA 3 -tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed ( t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above ( t = 120 min). The HA 3 -tagged Pol I subunit A43 was immunoprecipitated (α-HA-IPs) from 250 µl of extracts (Inputs) with anti-HA antibody. Fifty percent of the α-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3-ΔN-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantified using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. ( C ) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN), both expressing either chromosomally HA 3 -tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and sonified. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA 3 -tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of specific DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers specific for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. ( D ) Reduction of 35S pre-rRNA synthesis is attenuated in the ΔN-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were cultured to mid-log phase ( t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 µCi of [ 5 , 6- 3 H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3 H-labelled RNAs were visualized and quantified using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32 P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantified as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the different rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100.
Figure Legend Snippet: Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. ( A ) Gelfiltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were grown in YPD at 30°C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 µg) were separated on a Superose-6® column in a buffer containing 1.5 M potassium acetate. An amount of 250 µl of the collected 500 µl fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 µg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel filtration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. ( B ) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 (ΔN), both expressing chromosomally HA 3 -tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed ( t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above ( t = 120 min). The HA 3 -tagged Pol I subunit A43 was immunoprecipitated (α-HA-IPs) from 250 µl of extracts (Inputs) with anti-HA antibody. Fifty percent of the α-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3-ΔN-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantified using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. ( C ) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN), both expressing either chromosomally HA 3 -tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and sonified. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA 3 -tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of specific DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers specific for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. ( D ) Reduction of 35S pre-rRNA synthesis is attenuated in the ΔN-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were cultured to mid-log phase ( t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 µCi of [ 5 , 6- 3 H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3 H-labelled RNAs were visualized and quantified using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32 P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantified as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the different rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100.

Techniques Used: Cell Culture, Centrifugation, Lysis, Western Blot, Filtration, Expressing, Immunoprecipitation, Imaging, Software, Chromatin Immunoprecipitation, DNA Extraction, Real-time Polymerase Chain Reaction, Mutagenesis, Isolation, Agarose Gel Electrophoresis, Northern Blot

38) Product Images from "Cyclin-dependent kinase 5-mediated phosphorylation of chloride intracellular channel 4 promotes oxidative stress-induced neuronal death"

Article Title: Cyclin-dependent kinase 5-mediated phosphorylation of chloride intracellular channel 4 promotes oxidative stress-induced neuronal death

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0983-1

CLIC4 interacts with CDK5/p35. a Immunoblotting of proteins pulled down by glutathione Sepharose from lysates of N2a cells transiently transfected with GST alone or GST-CLIC4 as indicated. Membranes were probed with antibodies against the indicated proteins. b Immunoblotting of proteins pulled down by glutathione Sepharose from lysates of N2a cells transiently transfected with HA-CLIC4 and GST alone or GST-CLIC4. Membranes were probed with antibodies to the indicated proteins. c Immunoblotting of proteins immunoprecipitated from brain homogenates of wild-type C57BL/6 mice with antibodies against normal IgG or anti-CLIC4 antibody. d Immunoblotting of proteins immunoprecipitated from brain homogenates of wild-type C57BL/6 mice with antibodies against normal IgG, anti-CDK5 or anti-p35 antibody. e – g Immunofluorescence staining of DIV7 primary cortical neurons with anti-CDK5 and anti-CLIC4 antibodies showed the co-localization of CDK5 and CLIC4. Higher magnification around neuronal soma ( f ) and neurite ( g ) of the indicated area of e was presented and the co-localization was pointed with arrows. DAPI is a nucleus dye. Scale bar = 10 μm. h BiFC fluorescence and immunostaining showed that CDK5 and CLIC4 interacted in the cytoplasm of N2a cells. N2a cells were transfected with VN173-CDK5 and VC155 vector or VC155-CLIC4, then immunostained with anti-GFP antibody. DAPI is a nucleus dye. Scale bar = 10 μm
Figure Legend Snippet: CLIC4 interacts with CDK5/p35. a Immunoblotting of proteins pulled down by glutathione Sepharose from lysates of N2a cells transiently transfected with GST alone or GST-CLIC4 as indicated. Membranes were probed with antibodies against the indicated proteins. b Immunoblotting of proteins pulled down by glutathione Sepharose from lysates of N2a cells transiently transfected with HA-CLIC4 and GST alone or GST-CLIC4. Membranes were probed with antibodies to the indicated proteins. c Immunoblotting of proteins immunoprecipitated from brain homogenates of wild-type C57BL/6 mice with antibodies against normal IgG or anti-CLIC4 antibody. d Immunoblotting of proteins immunoprecipitated from brain homogenates of wild-type C57BL/6 mice with antibodies against normal IgG, anti-CDK5 or anti-p35 antibody. e – g Immunofluorescence staining of DIV7 primary cortical neurons with anti-CDK5 and anti-CLIC4 antibodies showed the co-localization of CDK5 and CLIC4. Higher magnification around neuronal soma ( f ) and neurite ( g ) of the indicated area of e was presented and the co-localization was pointed with arrows. DAPI is a nucleus dye. Scale bar = 10 μm. h BiFC fluorescence and immunostaining showed that CDK5 and CLIC4 interacted in the cytoplasm of N2a cells. N2a cells were transfected with VN173-CDK5 and VC155 vector or VC155-CLIC4, then immunostained with anti-GFP antibody. DAPI is a nucleus dye. Scale bar = 10 μm

Techniques Used: Transfection, Immunoprecipitation, Mouse Assay, Immunofluorescence, Staining, Bimolecular Fluorescence Complementation Assay, Fluorescence, Immunostaining, Plasmid Preparation

CDK5 promotes CLIC4 protein stability. a , b Protein levels of CLIC4 in N2a cells transiently transfected with GFP tag or GFP-p25 for 24 h. Relative CLIC4 levels were quantified in b ( n = 3 experiments). c , d CLIC4 and CDK5 protein levels in the brain homogenates of wild-type and CDK5−/− littermate embryos at E16.5. Embryos were genotyped and brain samples were analyzed by western blotting ( c ). Upper band in the agarose gel indicates CDK5−/− genotype, while lower one means wild-type. Relative CLIC4 levels were quantified in D ( n = 3 experiments, 4 embryos each group). e , f Immunoblotting of lysates of primary cortical neurons treated with 5 μM Roscovitine for 24 h. Relative CLIC4 levels were quantified in f ( n = 3 experiments). g , h Turnover of myc-CLIC4 and myc-CLIC4 (S108D) in N2a cells treated with 100 μM cycloheximide (CHX) for indicated times. Cell lysates were immunoblotted with anti-myc and anti-β-actin antibodies ( g ). Normalized myc-tagged CLIC4 proteins levels were quantified in h ( n = 3 experiments). i , j Turnover of HA-CLIC4 in N2a cells transiently transfected with HA-CLIC4/GFP-CDK5/GFP-p25 or HA-CLIC4/GFP-CDK5-KD/GFP-p25 and treated with CHX for indicated times. Lysates were subjected to western blotting for HA-tagged and GFP-tagged proteins and β-actin ( i ). Normalized HA-CLIC4 protein levels were quantified in j ( n = 3 experiments). Data are presented as the mean and SEM, and were analyzed by unpaired Student’s t -test ( b , d , f ) or two-way ANOVA test ( h , j ). * P
Figure Legend Snippet: CDK5 promotes CLIC4 protein stability. a , b Protein levels of CLIC4 in N2a cells transiently transfected with GFP tag or GFP-p25 for 24 h. Relative CLIC4 levels were quantified in b ( n = 3 experiments). c , d CLIC4 and CDK5 protein levels in the brain homogenates of wild-type and CDK5−/− littermate embryos at E16.5. Embryos were genotyped and brain samples were analyzed by western blotting ( c ). Upper band in the agarose gel indicates CDK5−/− genotype, while lower one means wild-type. Relative CLIC4 levels were quantified in D ( n = 3 experiments, 4 embryos each group). e , f Immunoblotting of lysates of primary cortical neurons treated with 5 μM Roscovitine for 24 h. Relative CLIC4 levels were quantified in f ( n = 3 experiments). g , h Turnover of myc-CLIC4 and myc-CLIC4 (S108D) in N2a cells treated with 100 μM cycloheximide (CHX) for indicated times. Cell lysates were immunoblotted with anti-myc and anti-β-actin antibodies ( g ). Normalized myc-tagged CLIC4 proteins levels were quantified in h ( n = 3 experiments). i , j Turnover of HA-CLIC4 in N2a cells transiently transfected with HA-CLIC4/GFP-CDK5/GFP-p25 or HA-CLIC4/GFP-CDK5-KD/GFP-p25 and treated with CHX for indicated times. Lysates were subjected to western blotting for HA-tagged and GFP-tagged proteins and β-actin ( i ). Normalized HA-CLIC4 protein levels were quantified in j ( n = 3 experiments). Data are presented as the mean and SEM, and were analyzed by unpaired Student’s t -test ( b , d , f ) or two-way ANOVA test ( h , j ). * P

Techniques Used: Transfection, Western Blot, Agarose Gel Electrophoresis

39) Product Images from "Multiple Interactions Drive Adaptor-Mediated Recruitment of the Ubiquitin Ligase Rsp5 to Membrane Proteins In Vivo and In Vitro"

Article Title: Multiple Interactions Drive Adaptor-Mediated Recruitment of the Ubiquitin Ligase Rsp5 to Membrane Proteins In Vivo and In Vitro

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E07-01-0011

Mutation of sequences within the cytoplasmic domain of Bsd2. (A) Growth of a Δ bsd2 strain in the presence of 25 μM CdCl 2 , complemented with either control vector (−), wild-type BSD2 ( BSD2 ), Y140A (ΔPY1), D146A (146), M147A (147), P149A (149), S150A (150), Y151A (151), Y152A (152), Y140A/P149A (ΔPY1,149), G170A/V173A/G174A (GVG), or D113A/G114A/V115A/F116A/S117A/N118A (113-8) BSD2 mutants. (B) Localization of GFP-Smf1ΔN and (C) GFP-Cps1 in a Δ bsd2 yeast strain complemented with wild-type (WT), control plasmid (ΔBsd2), or plasmids expressing D146A (146), M147A (147), P149A (149), S150A (150), Y151A (151), or Y152A (152) forms of Bsd 2 . (D) Localization of GFP-Tre1 and GFP-Cps1 in a Δ bsd2 strain expressing 113-8 Bsd2. (E) Localization in a Δ bsd2 Δ tul1 strain of GFP tagged wild-type Bsd2 (WT), P149A (149), Y151A (151) G170A/V173A/G174A (GVG), or D113A/G114A/V115A/F116A/S117A/N118A (113-8) Bsd2 mutants. (F) Immunoprecipitation of Triple HA-tagged Bsd 2 mutant proteins with protein-A tagged Rsp5 from Δ bsd2 Δ pep4 cells. IgG-Sepharose eluates were enriched over the extracts by a factor of 20.
Figure Legend Snippet: Mutation of sequences within the cytoplasmic domain of Bsd2. (A) Growth of a Δ bsd2 strain in the presence of 25 μM CdCl 2 , complemented with either control vector (−), wild-type BSD2 ( BSD2 ), Y140A (ΔPY1), D146A (146), M147A (147), P149A (149), S150A (150), Y151A (151), Y152A (152), Y140A/P149A (ΔPY1,149), G170A/V173A/G174A (GVG), or D113A/G114A/V115A/F116A/S117A/N118A (113-8) BSD2 mutants. (B) Localization of GFP-Smf1ΔN and (C) GFP-Cps1 in a Δ bsd2 yeast strain complemented with wild-type (WT), control plasmid (ΔBsd2), or plasmids expressing D146A (146), M147A (147), P149A (149), S150A (150), Y151A (151), or Y152A (152) forms of Bsd 2 . (D) Localization of GFP-Tre1 and GFP-Cps1 in a Δ bsd2 strain expressing 113-8 Bsd2. (E) Localization in a Δ bsd2 Δ tul1 strain of GFP tagged wild-type Bsd2 (WT), P149A (149), Y151A (151) G170A/V173A/G174A (GVG), or D113A/G114A/V115A/F116A/S117A/N118A (113-8) Bsd2 mutants. (F) Immunoprecipitation of Triple HA-tagged Bsd 2 mutant proteins with protein-A tagged Rsp5 from Δ bsd2 Δ pep4 cells. IgG-Sepharose eluates were enriched over the extracts by a factor of 20.

Techniques Used: Mutagenesis, Plasmid Preparation, Expressing, Immunoprecipitation

Sequences in Bsd2 adjacent to the membrane are involved in substrate recognition. (A) Localization of GFP-Cps1 and GFP-Tre1, with corresponding differential interference contrast images, in a Δ bsd2 yeast strain complemented with either wild-type (WT) or Bsd2 carrying G170A/V173A/G174A mutations (GVG). (B) Western blot of total protein extract from Δ bsd2 Δ tul1 cells expressing GFP-tagged Tlg1 CC205/6SS [GFP-Tlg1(SS)] in the presence of either a control plasmid (−), wild-type Bsd2 (WT), or Bsd2 G170A/V173A/G174A (GVG) driven from the BSD2 promoter. The blot was probed with an anti-GFP monoclonal antibody. (C) Protein A pull-down by tagged Tre1 (Δlumenal domain) of wild-type (WT), G170A/V173A/G174A (GVG), 113-8A (113-8), and ΔPY1 ΔPY2 (ΔPY12) triple HA-tagged Bsd2 from Δ bsd2 Δ pep4 cells. Eluates from IgG-Sepharose beads are enriched over the extracts by a factor of 80.
Figure Legend Snippet: Sequences in Bsd2 adjacent to the membrane are involved in substrate recognition. (A) Localization of GFP-Cps1 and GFP-Tre1, with corresponding differential interference contrast images, in a Δ bsd2 yeast strain complemented with either wild-type (WT) or Bsd2 carrying G170A/V173A/G174A mutations (GVG). (B) Western blot of total protein extract from Δ bsd2 Δ tul1 cells expressing GFP-tagged Tlg1 CC205/6SS [GFP-Tlg1(SS)] in the presence of either a control plasmid (−), wild-type Bsd2 (WT), or Bsd2 G170A/V173A/G174A (GVG) driven from the BSD2 promoter. The blot was probed with an anti-GFP monoclonal antibody. (C) Protein A pull-down by tagged Tre1 (Δlumenal domain) of wild-type (WT), G170A/V173A/G174A (GVG), 113-8A (113-8), and ΔPY1 ΔPY2 (ΔPY12) triple HA-tagged Bsd2 from Δ bsd2 Δ pep4 cells. Eluates from IgG-Sepharose beads are enriched over the extracts by a factor of 80.

Techniques Used: Western Blot, Expressing, Plasmid Preparation

Requirements for ubiquitination of Tre1. (A) Immunoprecipitation of GFP-tagged Tre1Δlumenal or Tre1 Δlumenal PPAG with protein A-tagged Rsp5 from Δ pep4 cells. Eluates from IgG-Sepharose beads enriched by a factor of 20. (B) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant Tre1Δlumenal (and Tre1 Δlumenal PPAG) by wild-type recombinant Rsp5 (WT) or Rsp5 carrying ΔWW1, ΔWW2, ΔWW3, ΔWW1,2, ΔWW1,3, or ΔWW2,3 mutations described above. (C) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant S-tagged TreΔlumenal PPAG by Rsp5 and Tap-purified wild-type Bsd2 or Tap-purified Bsd2 carrying ΔPY1, ΔPY2, 113-8, or GVG mutations described above. Arrows indicate mono-ubiquitinated forms of TreΔlumenal PPAG. (D) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant S-tagged TreΔlumenal PPAG mediated by Tap-purified Bsd2 and either wild-type Rsp5 or Rsp5 carrying ΔWW1, ΔWW2, ΔWW3, ΔWW1,2, ΔWW1,3, ΔWW2,3, ΔC2 mutations described above. Arrows indicate monoubiquitinated forms of TreΔlumenal PPAG. Irrelevant lanes in C and D have been excised, as indicated by the gaps, but exposures are identical for all lanes.
Figure Legend Snippet: Requirements for ubiquitination of Tre1. (A) Immunoprecipitation of GFP-tagged Tre1Δlumenal or Tre1 Δlumenal PPAG with protein A-tagged Rsp5 from Δ pep4 cells. Eluates from IgG-Sepharose beads enriched by a factor of 20. (B) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant Tre1Δlumenal (and Tre1 Δlumenal PPAG) by wild-type recombinant Rsp5 (WT) or Rsp5 carrying ΔWW1, ΔWW2, ΔWW3, ΔWW1,2, ΔWW1,3, or ΔWW2,3 mutations described above. (C) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant S-tagged TreΔlumenal PPAG by Rsp5 and Tap-purified wild-type Bsd2 or Tap-purified Bsd2 carrying ΔPY1, ΔPY2, 113-8, or GVG mutations described above. Arrows indicate mono-ubiquitinated forms of TreΔlumenal PPAG. (D) Western blot, probed with anti-S-tag, of in vitro ubiquitination of recombinant S-tagged TreΔlumenal PPAG mediated by Tap-purified Bsd2 and either wild-type Rsp5 or Rsp5 carrying ΔWW1, ΔWW2, ΔWW3, ΔWW1,2, ΔWW1,3, ΔWW2,3, ΔC2 mutations described above. Arrows indicate monoubiquitinated forms of TreΔlumenal PPAG. Irrelevant lanes in C and D have been excised, as indicated by the gaps, but exposures are identical for all lanes.

Techniques Used: Immunoprecipitation, Western Blot, In Vitro, Recombinant, Purification

40) Product Images from "Ubiquitination and dynactin regulate TMEPAI lysosomal trafficking"

Article Title: Ubiquitination and dynactin regulate TMEPAI lysosomal trafficking

Journal: Scientific Reports

doi: 10.1038/srep42668

Ubiquitin binding proteins Hrs and STAM regulate TMEPAI lysosomal trafficking. ( A ) Human Nedd4, Hrs, STAM, GGA3, Tsg101 and Eap45 were fused to the GAL4 activation domain (AD fusions) and tested for interaction with the TMEPAI cytoplasmic domain (aa63-287) fused to the GAL4 DNA-binding domain (BD fusion) in the yeast two-hybrid system. Yeast diploids co-expressing GAL4 AD fusions and GAL4 BD fusions were grown in liquid selective media, diploids were titrated (5 −1 , 5 −2 , 5 −3 ; total 3 μl) and patched on the DDO, QDO and QDO/X/A plates. Interaction between the indicated proteins results in growth on the QDO and QDO/X/A media. The yeast co-expressing TMEPAI and Nedd4 was used as the positive control. ( B ) HeLa cell lines co-transformed with pEF-IRES-TMEPAI-WT-Flag and pcDNA3.1 + -HA-Hrs or pcDNA3.1 + -HA-STAM were extracted and immunoprecipitated with antibody to Flag-tag. The immunoprecipitates were analyzed by Western blot with antibodies to HA and Flag. ( C ) GST, GST-tagged cytoplasmic domain of TMEPAI (GST-TMEPAI) and ubiquitinated GST-TMEPAI (GST-TMEPAI (Ub)) were coupled to Glutathione Sepharose beads and incubated with extracts from HeLa cells transiently transfected with pcDNA3.1 + -HA-Hrs or pcDNA3.1 + -HA-STAM. Bound proteins were detected by Western blot with antibodies to HA (left). Protein ubiquitination was analyzed by Western blot with antibody against TMEPAI (right). ( D ) Immunofluorescence microscopy of A549 cell lines infected with Hrs-shRNA (Hrs-KD) lentivirus 4 days before being fixed and double labelled with antibodies to TMEPAI and either WGA-FITC, Golgin84, Lamp2 and EEA1 (early endosome marker). Scrambled shRNA was used as negative control. Scale bar, 10 μM. Hrs-shRNA lentivirus efficiency was accessed by Western blot with antibody against Hrs and quantitation (N = 3, **P
Figure Legend Snippet: Ubiquitin binding proteins Hrs and STAM regulate TMEPAI lysosomal trafficking. ( A ) Human Nedd4, Hrs, STAM, GGA3, Tsg101 and Eap45 were fused to the GAL4 activation domain (AD fusions) and tested for interaction with the TMEPAI cytoplasmic domain (aa63-287) fused to the GAL4 DNA-binding domain (BD fusion) in the yeast two-hybrid system. Yeast diploids co-expressing GAL4 AD fusions and GAL4 BD fusions were grown in liquid selective media, diploids were titrated (5 −1 , 5 −2 , 5 −3 ; total 3 μl) and patched on the DDO, QDO and QDO/X/A plates. Interaction between the indicated proteins results in growth on the QDO and QDO/X/A media. The yeast co-expressing TMEPAI and Nedd4 was used as the positive control. ( B ) HeLa cell lines co-transformed with pEF-IRES-TMEPAI-WT-Flag and pcDNA3.1 + -HA-Hrs or pcDNA3.1 + -HA-STAM were extracted and immunoprecipitated with antibody to Flag-tag. The immunoprecipitates were analyzed by Western blot with antibodies to HA and Flag. ( C ) GST, GST-tagged cytoplasmic domain of TMEPAI (GST-TMEPAI) and ubiquitinated GST-TMEPAI (GST-TMEPAI (Ub)) were coupled to Glutathione Sepharose beads and incubated with extracts from HeLa cells transiently transfected with pcDNA3.1 + -HA-Hrs or pcDNA3.1 + -HA-STAM. Bound proteins were detected by Western blot with antibodies to HA (left). Protein ubiquitination was analyzed by Western blot with antibody against TMEPAI (right). ( D ) Immunofluorescence microscopy of A549 cell lines infected with Hrs-shRNA (Hrs-KD) lentivirus 4 days before being fixed and double labelled with antibodies to TMEPAI and either WGA-FITC, Golgin84, Lamp2 and EEA1 (early endosome marker). Scrambled shRNA was used as negative control. Scale bar, 10 μM. Hrs-shRNA lentivirus efficiency was accessed by Western blot with antibody against Hrs and quantitation (N = 3, **P

Techniques Used: Binding Assay, Activation Assay, Expressing, Positive Control, Transformation Assay, Immunoprecipitation, FLAG-tag, Western Blot, Incubation, Transfection, Immunofluorescence, Microscopy, Infection, shRNA, Whole Genome Amplification, Marker, Negative Control, Quantitation Assay

TMEPAI interacts with dynactin 5 and dynactin 6. ( A ) TMEPAI cytoplasmic domain (aa63-287) was tested for interaction with dynactin complex subunits dynactin 4 (DCTN4), dynactin 5 (DCTN5), dynactin 6 (DCTN6) and Arp1 using the yeast two-hybrid system. Yeast diploids were grown in liquid selective media. Diploids were titrated (5-1, 5-2, 5-3; total 3 μl) and patched on the DDO, QDO and QDO/X/A plates. The yeast co-expressing TMEPAI and Nedd4 was used as the positive control. ( B ) GST and the GST-tagged cytoplasmic domain of TMEPAI (GST-TMEPAI) were coupled to Glutathione Sepharose beads and incubated with His-HA-tagged recombinant proteins DCTN5 and DCTN6. Bound proteins were detected by Western blot with antibody to HA (left). GST, GST-TMEPAI proteins used for pull-down assay was analyzed by Western blot with antibody against GST (right). ( C ) Glutathione beads loaded with recombinant GST fused TMEPAI were incubated with His-HA-tagged recombinant DCTN4, DCTN5, DCTN6. The His-HA-DCTN4/5/6 in the reaction mixtures (input) and those bound to the GST-TMEPAI coupled beads (Pull down) were determined by Western blot using anti-HA antibody.
Figure Legend Snippet: TMEPAI interacts with dynactin 5 and dynactin 6. ( A ) TMEPAI cytoplasmic domain (aa63-287) was tested for interaction with dynactin complex subunits dynactin 4 (DCTN4), dynactin 5 (DCTN5), dynactin 6 (DCTN6) and Arp1 using the yeast two-hybrid system. Yeast diploids were grown in liquid selective media. Diploids were titrated (5-1, 5-2, 5-3; total 3 μl) and patched on the DDO, QDO and QDO/X/A plates. The yeast co-expressing TMEPAI and Nedd4 was used as the positive control. ( B ) GST and the GST-tagged cytoplasmic domain of TMEPAI (GST-TMEPAI) were coupled to Glutathione Sepharose beads and incubated with His-HA-tagged recombinant proteins DCTN5 and DCTN6. Bound proteins were detected by Western blot with antibody to HA (left). GST, GST-TMEPAI proteins used for pull-down assay was analyzed by Western blot with antibody against GST (right). ( C ) Glutathione beads loaded with recombinant GST fused TMEPAI were incubated with His-HA-tagged recombinant DCTN4, DCTN5, DCTN6. The His-HA-DCTN4/5/6 in the reaction mixtures (input) and those bound to the GST-TMEPAI coupled beads (Pull down) were determined by Western blot using anti-HA antibody.

Techniques Used: Expressing, Positive Control, Incubation, Recombinant, Western Blot, Pull Down Assay

41) Product Images from "The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases"

Article Title: The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases

Journal: PLoS ONE

doi: 10.1371/journal.pone.0161672

PDRG1 interacts with methionine adenosyltransferase α1. (A) Growth of yeast cotransfectants harboring pGBKT7-MATα1 (bait) and pACT2 plasmids (prey) including ORFs of MATα1, PDRG1, clone M2, clone M6 or laminin (negative control) in low (-LW) and high (-AHLW) stringency SC media. Additional controls including the empty pGBK plasmid are shown on the right. (B) Representative anti-FLAG immunoprecipitation results from four independent experiments using total lysates of CHO cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). The size of the standards is indicated on the left side of the panel. (C) Representative anti-HA immunoprecipitation data from three independent experiments utilizing total lysates of HEK 293T cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). Western blots of the input fractions were developed using anti-FLAG and anti-HA, whereas immunoprecipitates were analyzed using anti-HA or anti-FLAG with mouse TrueBlot ULTRA, as required. The arrow indicates an unspecific band recognized by anti-FLAG slightly over the FLAG-MATα1 signal in HEK 293T samples. The size of the standards is indicated on the left side of the panel. (D) Pull-down confirmation of the interaction using glutathione Sepharose beads loaded with GST or GST-PDRG1 and incubated with recombinant MATα1 plus excess GST. Results shown correspond to a typical experiments out of the five carried out; input fractions of the recombinant proteins used (left) and pull-down results (right) are shown. The size of the standards is indicated on the left side of the panel.
Figure Legend Snippet: PDRG1 interacts with methionine adenosyltransferase α1. (A) Growth of yeast cotransfectants harboring pGBKT7-MATα1 (bait) and pACT2 plasmids (prey) including ORFs of MATα1, PDRG1, clone M2, clone M6 or laminin (negative control) in low (-LW) and high (-AHLW) stringency SC media. Additional controls including the empty pGBK plasmid are shown on the right. (B) Representative anti-FLAG immunoprecipitation results from four independent experiments using total lysates of CHO cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). The size of the standards is indicated on the left side of the panel. (C) Representative anti-HA immunoprecipitation data from three independent experiments utilizing total lysates of HEK 293T cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). Western blots of the input fractions were developed using anti-FLAG and anti-HA, whereas immunoprecipitates were analyzed using anti-HA or anti-FLAG with mouse TrueBlot ULTRA, as required. The arrow indicates an unspecific band recognized by anti-FLAG slightly over the FLAG-MATα1 signal in HEK 293T samples. The size of the standards is indicated on the left side of the panel. (D) Pull-down confirmation of the interaction using glutathione Sepharose beads loaded with GST or GST-PDRG1 and incubated with recombinant MATα1 plus excess GST. Results shown correspond to a typical experiments out of the five carried out; input fractions of the recombinant proteins used (left) and pull-down results (right) are shown. The size of the standards is indicated on the left side of the panel.

Techniques Used: Negative Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Incubation, Recombinant

Pull-down analysis of PDRG1 interaction with MATα2 and MAT II. (A) Representative western blots of pull-down experiments using glutathione Sepharose beads loaded with GST or GST-PDRG1 and recombinant MATα2, MATβ or the hetero-oligomer MAT II; anti-GST, anti-MATα2 and MATβ were used for detection. The size of the standards is indicated on the left side of the panels. (B) Quantification of the MATα2/GST-PDRG1 signal ratio (mean ± SEM) from five independent pull-down experiments. (C) Representative western blots of pull-down experiments carried out with the truncated PDRG1 forms and recombinant MATα2 using anti-GST and anti-MATα2. The size of the standards is indicated on the left side of the panels. (D) Quantification of the MATα2/GST-PDRG1 signal ratio (mean ± SEM) from five independent pull-down experiments. All the incubations with MAT subunits or MAT II were carried out in the presence of excess GST to avoid unspecific binding. (*p≤0.05 vs GST-PDRG1).
Figure Legend Snippet: Pull-down analysis of PDRG1 interaction with MATα2 and MAT II. (A) Representative western blots of pull-down experiments using glutathione Sepharose beads loaded with GST or GST-PDRG1 and recombinant MATα2, MATβ or the hetero-oligomer MAT II; anti-GST, anti-MATα2 and MATβ were used for detection. The size of the standards is indicated on the left side of the panels. (B) Quantification of the MATα2/GST-PDRG1 signal ratio (mean ± SEM) from five independent pull-down experiments. (C) Representative western blots of pull-down experiments carried out with the truncated PDRG1 forms and recombinant MATα2 using anti-GST and anti-MATα2. The size of the standards is indicated on the left side of the panels. (D) Quantification of the MATα2/GST-PDRG1 signal ratio (mean ± SEM) from five independent pull-down experiments. All the incubations with MAT subunits or MAT II were carried out in the presence of excess GST to avoid unspecific binding. (*p≤0.05 vs GST-PDRG1).

Techniques Used: Western Blot, Recombinant, Binding Assay

42) Product Images from "Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity *"

Article Title: Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.368654

STEP dephosphorylates Pyk2 at Tyr 402 . A , Pyk2 was immunoprecipitated from mouse brain lysates using anti-Pyk2 antibody and protein A/G-agarose in the presence of pervanadate ( Perv ) (1 m m ). Mg-ATP, STEP (10 μ m ), and pervanadate (1 m m ) were added
Figure Legend Snippet: STEP dephosphorylates Pyk2 at Tyr 402 . A , Pyk2 was immunoprecipitated from mouse brain lysates using anti-Pyk2 antibody and protein A/G-agarose in the presence of pervanadate ( Perv ) (1 m m ). Mg-ATP, STEP (10 μ m ), and pervanadate (1 m m ) were added

Techniques Used: Immunoprecipitation

Phosphorylation of Pyk2 at Tyr 402 is increased in presence of protein-tyrosine phosphatase inhibitor. A , rat brain extracts were incubated with 1 μg of αN-Pyk2 antibody in the absence of protein A-agarose. Mg-ATP was then added in the
Figure Legend Snippet: Phosphorylation of Pyk2 at Tyr 402 is increased in presence of protein-tyrosine phosphatase inhibitor. A , rat brain extracts were incubated with 1 μg of αN-Pyk2 antibody in the absence of protein A-agarose. Mg-ATP was then added in the

Techniques Used: Incubation

43) Product Images from "Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2"

Article Title: Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2

Journal: Scientific Reports

doi: 10.1038/srep43471

DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) HEK-293 cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
Figure Legend Snippet: DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) HEK-293 cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1

Techniques Used: In Vitro, In Vivo, Incubation, Western Blot, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Immunoprecipitation

DUSP2 dephosphorylates Serine 189 and 186 in the activation loop of ERK3 and ERK4 respectively in vivo . ( a ) HeLa cells were co-transfected with expression vectors encoding a kinase-dead mutant of ERK4 fused to green fluorescent protein (GFP-ERK4D168A) and either myc-tagged DUSP2, MKP-1, or MKP-3. After 24 h, whole cell extracts were prepared and GFP-ERK4D168A was immunoprecipitated using an anti-GFP antibody. The phosphorylation status of Serine 186 within the activation loop in immunoprecipitated ERK4 D168A was then analysed by Western blotting using an anti-phospho S186 antibody. The levels of GFP-ERK4D168A in immunoprecipitates and of the three MKPs in whole cell extracts were analysed by Western blotting using antibodies against ERK4 and myc, respectively. ( b ) HeLa cells were co-transfected with vectors encoding either myc-tagged ERK4 or ERK4D320N together with either HA-tagged DUSP2, DUSP2KIM, or DUSP2CS. After 24 h, whole cell extracts were prepared and either ERK4 or ERK4D320N were immunoprecipitated using an anti-myc antibody. The phosphorylation status of Serine 186 was analysed as described in A. Co-immunoprecipitated DUSP2 was visualized using an anti-HA antibody. The levels of overexpressed ERK4- and DUSP2 proteins were analysed by Western blotting of whole cell extracts using anti-myc and anti–HA antibodies, respectively. ( c ) NCI-H1299 cells were co-transfected with expression vectors encoding a Flag-tagged kinase-dead mutant of ERK3 (ERK3D171A) and either an empty expression vector or plasmids encoding wild-type DUSP2, a catalytically inactive DUSP2 mutant (DUSP2CS) or a kinase interaction motif-deficient mutant (DUSP2KIM) respectively. After 24 h, cell extracts were prepared and ERK3 was immunoprecipitated using M2-FLAG conjugated agarose. The Phosphorylation status of Serine 189 within the activation loop of immunoprecipitated ERK3 was analysed by Western-blotting using a specific anti-phospho Serine 189 ERK3 antibody. Levels of ERK3 protein in the input lysates and immunoprecipitates were analysed by Western-blotting using an anti-FLAG antibody. The expression of myc-tagged DUSP2 proteins in cell lysates and immunoprecipitates were analysed by Western-blotting using an anti-myc or anti-DUSP2 antibody respectively. All experiments were performed 3 times and representative images are shown. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
Figure Legend Snippet: DUSP2 dephosphorylates Serine 189 and 186 in the activation loop of ERK3 and ERK4 respectively in vivo . ( a ) HeLa cells were co-transfected with expression vectors encoding a kinase-dead mutant of ERK4 fused to green fluorescent protein (GFP-ERK4D168A) and either myc-tagged DUSP2, MKP-1, or MKP-3. After 24 h, whole cell extracts were prepared and GFP-ERK4D168A was immunoprecipitated using an anti-GFP antibody. The phosphorylation status of Serine 186 within the activation loop in immunoprecipitated ERK4 D168A was then analysed by Western blotting using an anti-phospho S186 antibody. The levels of GFP-ERK4D168A in immunoprecipitates and of the three MKPs in whole cell extracts were analysed by Western blotting using antibodies against ERK4 and myc, respectively. ( b ) HeLa cells were co-transfected with vectors encoding either myc-tagged ERK4 or ERK4D320N together with either HA-tagged DUSP2, DUSP2KIM, or DUSP2CS. After 24 h, whole cell extracts were prepared and either ERK4 or ERK4D320N were immunoprecipitated using an anti-myc antibody. The phosphorylation status of Serine 186 was analysed as described in A. Co-immunoprecipitated DUSP2 was visualized using an anti-HA antibody. The levels of overexpressed ERK4- and DUSP2 proteins were analysed by Western blotting of whole cell extracts using anti-myc and anti–HA antibodies, respectively. ( c ) NCI-H1299 cells were co-transfected with expression vectors encoding a Flag-tagged kinase-dead mutant of ERK3 (ERK3D171A) and either an empty expression vector or plasmids encoding wild-type DUSP2, a catalytically inactive DUSP2 mutant (DUSP2CS) or a kinase interaction motif-deficient mutant (DUSP2KIM) respectively. After 24 h, cell extracts were prepared and ERK3 was immunoprecipitated using M2-FLAG conjugated agarose. The Phosphorylation status of Serine 189 within the activation loop of immunoprecipitated ERK3 was analysed by Western-blotting using a specific anti-phospho Serine 189 ERK3 antibody. Levels of ERK3 protein in the input lysates and immunoprecipitates were analysed by Western-blotting using an anti-FLAG antibody. The expression of myc-tagged DUSP2 proteins in cell lysates and immunoprecipitates were analysed by Western-blotting using an anti-myc or anti-DUSP2 antibody respectively. All experiments were performed 3 times and representative images are shown. Unprocessed original scans of the blots are shown in Supplementary Fig. 1

Techniques Used: Activation Assay, In Vivo, Transfection, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Plasmid Preparation

44) Product Images from "SCFSAP controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana"

Article Title: SCFSAP controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana

Journal: Nature Communications

doi: 10.1038/ncomms11192

SAP physically associates with components of the SCF complex. ( a ) The F-box motif of SAP is required for the interactions with ASK1 and ASK2 in yeast cells. The SAP protein contains a serine/glycine-rich domain, an F-box motif and a WD40-like domain. The indicated construct pairs were co-transformed into yeast strain Y2HGold (Clontech). Interactions between bait and prey were examined on the control media −2 (SD/-Leu/-Trp) and selective media −4 (SD/-Ade/-His/-Leu/-Trp). ( b ) SAP interacts with ASK1 and ASK2 in vitro . His-ASK1 and His-ASK2 were pulled down (PD) by GST-SAP immobilized on glutathione sepharose and analysed by immunoblotting (IB) using an anti-His antibody. The amount of GST-GUS or GST-SAP was visualized by Coomassie Brilliant Blue (CBB) staining. ( c ) SAP associates with ASK1 and ASK2 in vivo . N. benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harbouring 35S:GFP-ASK1/2 and 35S:Myc-SAP plasmids. Total proteins were immunoprecipitated with GFP-Trap-A and the immunoblot was probed with anti-GFP and anti-Myc antibodies, respectively. Myc-SAP was detected in the immunoprecipitated GFP-ASK1 and GFP-ASK2 complex. ( d ) SAP associates with CUL1 in vivo . N. benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harbouring 35S:GFP-SAP and 35S:Myc-CUL1 plasmids. Total proteins were immunoprecipitated with GFP-Trap-A and the immunoblot was probed with anti-GFP and anti-Myc antibodies, respectively. Myc-CUL1 was detected in the immunoprecipitated GFP-SAP complex.
Figure Legend Snippet: SAP physically associates with components of the SCF complex. ( a ) The F-box motif of SAP is required for the interactions with ASK1 and ASK2 in yeast cells. The SAP protein contains a serine/glycine-rich domain, an F-box motif and a WD40-like domain. The indicated construct pairs were co-transformed into yeast strain Y2HGold (Clontech). Interactions between bait and prey were examined on the control media −2 (SD/-Leu/-Trp) and selective media −4 (SD/-Ade/-His/-Leu/-Trp). ( b ) SAP interacts with ASK1 and ASK2 in vitro . His-ASK1 and His-ASK2 were pulled down (PD) by GST-SAP immobilized on glutathione sepharose and analysed by immunoblotting (IB) using an anti-His antibody. The amount of GST-GUS or GST-SAP was visualized by Coomassie Brilliant Blue (CBB) staining. ( c ) SAP associates with ASK1 and ASK2 in vivo . N. benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harbouring 35S:GFP-ASK1/2 and 35S:Myc-SAP plasmids. Total proteins were immunoprecipitated with GFP-Trap-A and the immunoblot was probed with anti-GFP and anti-Myc antibodies, respectively. Myc-SAP was detected in the immunoprecipitated GFP-ASK1 and GFP-ASK2 complex. ( d ) SAP associates with CUL1 in vivo . N. benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harbouring 35S:GFP-SAP and 35S:Myc-CUL1 plasmids. Total proteins were immunoprecipitated with GFP-Trap-A and the immunoblot was probed with anti-GFP and anti-Myc antibodies, respectively. Myc-CUL1 was detected in the immunoprecipitated GFP-SAP complex.

Techniques Used: Construct, Transformation Assay, In Vitro, Staining, In Vivo, Injection, Immunoprecipitation

SAP physically associates with and targets PPD proteins for degradation. ( a ) The bimolecular fluorescence complementation (BiFC) assays indicate that SAP interacts with PPD1 and PPD2 in N. benthamiana. nYFP-SAP and cYFP-PPD1/2 were coexpressed in leaves of N. benthamiana . DAPI staining indicates the nuclei. ( b ) SAP interacts with PPD1 and PPD2 in Arabidopsis . 35S:GFP-SAP;35S:Myc-PPD1 and 35S:GFP-SAP;35S:Myc-PPD2 transgenic Arabidopsis plants were used to perform coimmunoprecipitation. Total proteins from 35S:GFP;35S:Myc-PPD1 (1), 35S:GFP-SAP;35S:Myc-PPD1 (2), 35S:GFP;35S:Myc-PPD2 (3) and 35S:GFP-SAP;35S:Myc-PPD2 (4) leaves were isolated and incubated with GFP-Trap-A agarose beads and precipitates were detected with anti-GFP or anti-Myc antibodies, respectively. ( c ) The proteasome inhibitor MG132 stabilizes PPD1. Ten-day-old 35S:Myc-PPD1 seedlings were treated with or without 50 μM MG132. Total protein extracts were subjected to immunoblot assays using anti-Myc and anti-RPN6 (as loading control) antibodies. Quantification of Myc-PPD1 protein levels was relative to RPN6. ( d ) The proteasome inhibitor MG132 stabilizes PPD2. Ten-day-old 35S:Myc-PPD2 seedlings were treated with or without 50 μM MG132. Total protein extracts were subjected to immunoblot assays using anti-Myc and anti-RPN6 antibodies. Quantification of Myc-PPD2 protein levels was relative to RPN6. ( e ) Overexpression of SAP results in the reduced levels of PPD1 proteins. Total proteins from 35S:GFP;35S:Myc-PPD1 (1) and 35S:GFP-SAP;35S:Myc-PPD1 (2) leaves were isolated and subjected to immunoblot assays using anti-Myc and anti-RPN6 antibodies, respectively. Quantification of GFP-PPD1 protein levels was relative to RPN6. ( f ) Overexpression of SAP results in the reduced levels of PPD2 proteins. Total proteins from 35S:GFP;35S:Myc-PPD2 (3) and 35S:GFP-SAP;35S:Myc-PPD2 (4) leaves were isolated and subjected to immunoblot assays using anti-Myc and anti-RPN6 antibodies, respectively. Quantification of GFP-PPD2 protein levels was relative to RPN6. ( g ) The GFP-PPD1 proteins accumulate at higher levels in the sod3-1 mutant. Total proteins from 10-day-old 35S:GFP-PPD1 and 35S:GFP-PPD1;sod3-1 seedlings were subjected to immunoblot assays using anti-GFP and anti-RPN6 antibodies, respectively. Quantification of GFP-PPD1 protein levels was relative to RPN6. ( h ) The GFP-PPD2 proteins accumulate at higher levels in the sod3-1 mutant. Total proteins from 10-day-old 35S:GFP-PPD2 and 35S:GFP-PPD2;sod3-1 seedlings were subjected to immunoblot assays using anti-GFP and anti-RPN6 (as loading control) antibodies, respectively. Quantification of GFP-PPD2 protein levels was relative to RPN6. Scale bars, 50 μm ( a ).
Figure Legend Snippet: SAP physically associates with and targets PPD proteins for degradation. ( a ) The bimolecular fluorescence complementation (BiFC) assays indicate that SAP interacts with PPD1 and PPD2 in N. benthamiana. nYFP-SAP and cYFP-PPD1/2 were coexpressed in leaves of N. benthamiana . DAPI staining indicates the nuclei. ( b ) SAP interacts with PPD1 and PPD2 in Arabidopsis . 35S:GFP-SAP;35S:Myc-PPD1 and 35S:GFP-SAP;35S:Myc-PPD2 transgenic Arabidopsis plants were used to perform coimmunoprecipitation. Total proteins from 35S:GFP;35S:Myc-PPD1 (1), 35S:GFP-SAP;35S:Myc-PPD1 (2), 35S:GFP;35S:Myc-PPD2 (3) and 35S:GFP-SAP;35S:Myc-PPD2 (4) leaves were isolated and incubated with GFP-Trap-A agarose beads and precipitates were detected with anti-GFP or anti-Myc antibodies, respectively. ( c ) The proteasome inhibitor MG132 stabilizes PPD1. Ten-day-old 35S:Myc-PPD1 seedlings were treated with or without 50 μM MG132. Total protein extracts were subjected to immunoblot assays using anti-Myc and anti-RPN6 (as loading control) antibodies. Quantification of Myc-PPD1 protein levels was relative to RPN6. ( d ) The proteasome inhibitor MG132 stabilizes PPD2. Ten-day-old 35S:Myc-PPD2 seedlings were treated with or without 50 μM MG132. Total protein extracts were subjected to immunoblot assays using anti-Myc and anti-RPN6 antibodies. Quantification of Myc-PPD2 protein levels was relative to RPN6. ( e ) Overexpression of SAP results in the reduced levels of PPD1 proteins. Total proteins from 35S:GFP;35S:Myc-PPD1 (1) and 35S:GFP-SAP;35S:Myc-PPD1 (2) leaves were isolated and subjected to immunoblot assays using anti-Myc and anti-RPN6 antibodies, respectively. Quantification of GFP-PPD1 protein levels was relative to RPN6. ( f ) Overexpression of SAP results in the reduced levels of PPD2 proteins. Total proteins from 35S:GFP;35S:Myc-PPD2 (3) and 35S:GFP-SAP;35S:Myc-PPD2 (4) leaves were isolated and subjected to immunoblot assays using anti-Myc and anti-RPN6 antibodies, respectively. Quantification of GFP-PPD2 protein levels was relative to RPN6. ( g ) The GFP-PPD1 proteins accumulate at higher levels in the sod3-1 mutant. Total proteins from 10-day-old 35S:GFP-PPD1 and 35S:GFP-PPD1;sod3-1 seedlings were subjected to immunoblot assays using anti-GFP and anti-RPN6 antibodies, respectively. Quantification of GFP-PPD1 protein levels was relative to RPN6. ( h ) The GFP-PPD2 proteins accumulate at higher levels in the sod3-1 mutant. Total proteins from 10-day-old 35S:GFP-PPD2 and 35S:GFP-PPD2;sod3-1 seedlings were subjected to immunoblot assays using anti-GFP and anti-RPN6 (as loading control) antibodies, respectively. Quantification of GFP-PPD2 protein levels was relative to RPN6. Scale bars, 50 μm ( a ).

Techniques Used: Fluorescence, Bimolecular Fluorescence Complementation Assay, Staining, Transgenic Assay, Isolation, Incubation, Over Expression, Mutagenesis

45) Product Images from "Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis"

Article Title: Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis

Journal: Nature cell biology

doi: 10.1038/ncb1747

GAPDH interacts with p300/CBP and GAPDH-K160R acts as a dominant-negative mutant. ( a ) GAPDH, native or NO-modified, binds similarly in vitro to a fragment of human p300 (F-p300) (amino acids 1135−2414). GST or GST–GAPDH was pre-treated with 50 μM GSH or GSNO for 30 min at 37 °C. F-p300 was added and binding assessed by a GSH-agarose pulldown assay. ( b ) GAPDH–p300 binding occurs in RAW264.7 cells treated with LPS/IFNγ for 16 h and is abolished by the iNOS inhibitor 1400W (100 μM). Cell lysates were immunoprecipitated with an anti-p300 antibody and the immunoprecipitates were analysed by western blotting with an anti-GAPDH antibody. ( c ) GAPDH and p300 are colocalized in the nucleus of RAW264.7 cells after exposure to LPS/IFNγ. Cells were stained with immunofluorescent anti-GAPDH and anti-p300 antibodies (green, p300; red, GAPDH). Scale bar, 10 μm. ( d ) R -(−)-Deprenyl (Dep) inhibits the enhancement of GAPDH–p300/CBP binding in mouse brain elicited by MPTP treatment. ( e ) K160R mutation of GAPDH abolishes GAPDH–p300 interactions in HEK293 cells. ( f ) GAPDH-K160R expression prevents acetylation of endogenous GAPDH and its binding to p300/CBP in a concentration-dependent manner, suggesting that GAPDH-K160R functions as a dominant-negative mutant. Forty-eight hours after transfection with 0.1, 0.5, 2, 3, or 4.5 μg of HA–GAPDH-K160R, HEK293 cells were treated with 200 μM GSNO for 24 h. Cell lysates were immunoprecipitated with anti-GAPDH, anti-p300, anti-CBP or anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with anti-HA or anti-GAPDH antibodies. Arrows indicate endogenous GAPDH and arrowheads indicate exogenous HA–GAPDH-K160R. ( g ) Nuclear localization of GAPDH augments its binding to p300/CBP and acetylation at Lys 160. Peritoneal macrophages from wild-type and iNOS knockout mice were transfected with various GAPDH constructs. Cell lysates were immunoprecipitated with anti-p300, anti-CBP or anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with anti-HA antibody. Both HA–NLS–GAPDH and HA–NLS–GAPDH–C150S, but not HA–NLS–GAPDH-K160R, augmented GAPDH–p300/CBP binding, as well as GAPDH acetylation in both iNOS knockout and wild-type cells.
Figure Legend Snippet: GAPDH interacts with p300/CBP and GAPDH-K160R acts as a dominant-negative mutant. ( a ) GAPDH, native or NO-modified, binds similarly in vitro to a fragment of human p300 (F-p300) (amino acids 1135−2414). GST or GST–GAPDH was pre-treated with 50 μM GSH or GSNO for 30 min at 37 °C. F-p300 was added and binding assessed by a GSH-agarose pulldown assay. ( b ) GAPDH–p300 binding occurs in RAW264.7 cells treated with LPS/IFNγ for 16 h and is abolished by the iNOS inhibitor 1400W (100 μM). Cell lysates were immunoprecipitated with an anti-p300 antibody and the immunoprecipitates were analysed by western blotting with an anti-GAPDH antibody. ( c ) GAPDH and p300 are colocalized in the nucleus of RAW264.7 cells after exposure to LPS/IFNγ. Cells were stained with immunofluorescent anti-GAPDH and anti-p300 antibodies (green, p300; red, GAPDH). Scale bar, 10 μm. ( d ) R -(−)-Deprenyl (Dep) inhibits the enhancement of GAPDH–p300/CBP binding in mouse brain elicited by MPTP treatment. ( e ) K160R mutation of GAPDH abolishes GAPDH–p300 interactions in HEK293 cells. ( f ) GAPDH-K160R expression prevents acetylation of endogenous GAPDH and its binding to p300/CBP in a concentration-dependent manner, suggesting that GAPDH-K160R functions as a dominant-negative mutant. Forty-eight hours after transfection with 0.1, 0.5, 2, 3, or 4.5 μg of HA–GAPDH-K160R, HEK293 cells were treated with 200 μM GSNO for 24 h. Cell lysates were immunoprecipitated with anti-GAPDH, anti-p300, anti-CBP or anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with anti-HA or anti-GAPDH antibodies. Arrows indicate endogenous GAPDH and arrowheads indicate exogenous HA–GAPDH-K160R. ( g ) Nuclear localization of GAPDH augments its binding to p300/CBP and acetylation at Lys 160. Peritoneal macrophages from wild-type and iNOS knockout mice were transfected with various GAPDH constructs. Cell lysates were immunoprecipitated with anti-p300, anti-CBP or anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with anti-HA antibody. Both HA–NLS–GAPDH and HA–NLS–GAPDH–C150S, but not HA–NLS–GAPDH-K160R, augmented GAPDH–p300/CBP binding, as well as GAPDH acetylation in both iNOS knockout and wild-type cells.

Techniques Used: Dominant Negative Mutation, Modification, In Vitro, Binding Assay, Immunoprecipitation, Western Blot, Staining, Mutagenesis, Expressing, Concentration Assay, Transfection, Knock-Out, Mouse Assay, Construct

GAPDH–p300 activates downstream targets, such as p53 and PUMA. ( a ) p53 is acetylated in GSNO-treated HEK293 cells; this is abolished by expression of GAPDH-K160R. Cell lysates were immunoprecipitated with anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with an anti-p53 antibody. ( b ) GAPDH–p53 binding was augmented by treatment with LPS/IFNγ for 16 h in RAW 264.7 cells. This was blocked by 1400W (100 μM). Cell lysates were immunoprecipitated with an anti-p53 antibody and the immunoprecipitates were analysed by western blotting with an anti-GAPDH antibody. ( c ) GAPDH–p300–p53 forms a complex in vitro , with p300 required for the interaction. Binding was examined by GSH-agarose pulldown assay. ( d) Formation of a p53–p300-sulphonated GAPDH (sGAPDH) complex at the PUMA promoter region in U2OS cells treated with GSNO, assayed by ChIP assay. Cells were treated with 200 μM GSH or GSNO for 24 h. ( e ) Formation of p53–p300–sGAPDH complex at the PUMA promoter region in U2OS cells with GSNO was blocked by expression of GAPDH-K160R. Forty-eight hours after transfection with HA–GAPDH or HA–GAPDH-K160R, cells were treated with 200 μM GSNO for 24 h. ( f ) RNAi depletion of GAPDH diminishes the formation of the p53–p300– sGAPDH complex at the PUMA promoter in U2OS cells. Forty-eight hours after transfection with control or GAPDH siRNA, cells were treated with 200 μM GSH or GSNO for 24 h. * P
Figure Legend Snippet: GAPDH–p300 activates downstream targets, such as p53 and PUMA. ( a ) p53 is acetylated in GSNO-treated HEK293 cells; this is abolished by expression of GAPDH-K160R. Cell lysates were immunoprecipitated with anti-acetyl Lys antibody and the immunoprecipitates were analysed by western blotting with an anti-p53 antibody. ( b ) GAPDH–p53 binding was augmented by treatment with LPS/IFNγ for 16 h in RAW 264.7 cells. This was blocked by 1400W (100 μM). Cell lysates were immunoprecipitated with an anti-p53 antibody and the immunoprecipitates were analysed by western blotting with an anti-GAPDH antibody. ( c ) GAPDH–p300–p53 forms a complex in vitro , with p300 required for the interaction. Binding was examined by GSH-agarose pulldown assay. ( d) Formation of a p53–p300-sulphonated GAPDH (sGAPDH) complex at the PUMA promoter region in U2OS cells treated with GSNO, assayed by ChIP assay. Cells were treated with 200 μM GSH or GSNO for 24 h. ( e ) Formation of p53–p300–sGAPDH complex at the PUMA promoter region in U2OS cells with GSNO was blocked by expression of GAPDH-K160R. Forty-eight hours after transfection with HA–GAPDH or HA–GAPDH-K160R, cells were treated with 200 μM GSNO for 24 h. ( f ) RNAi depletion of GAPDH diminishes the formation of the p53–p300– sGAPDH complex at the PUMA promoter in U2OS cells. Forty-eight hours after transfection with control or GAPDH siRNA, cells were treated with 200 μM GSH or GSNO for 24 h. * P

Techniques Used: Expressing, Immunoprecipitation, Western Blot, Binding Assay, In Vitro, Chromatin Immunoprecipitation, Transfection

46) Product Images from "Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression"

Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1708427114

Santa Cruz sc-365536 mouse monoclonal antibody (sc) recognizes C-terminal TRPM6 fragments larger than ∼70 kDa. C-terminally HA-tagged TRPM6 stably expressed in 293T cells, immunoprecipitated with anti-HA agarose, and probed on Western blot with anti–HA-peroxidase or sc-365536. Thus, the sc-365536 epitope is located N-terminal to amino acid ∼1400.
Figure Legend Snippet: Santa Cruz sc-365536 mouse monoclonal antibody (sc) recognizes C-terminal TRPM6 fragments larger than ∼70 kDa. C-terminally HA-tagged TRPM6 stably expressed in 293T cells, immunoprecipitated with anti-HA agarose, and probed on Western blot with anti–HA-peroxidase or sc-365536. Thus, the sc-365536 epitope is located N-terminal to amino acid ∼1400.

Techniques Used: Stable Transfection, Immunoprecipitation, Western Blot

M6CK binds PRMT5, WDR77, and ICLN subunits of the protein methylase complex. ( A ) Endogenous TRPM6 binds endogenous PRMT5 and associated proteins. Extracts of 293T cells with endogenous HA-tagged TRPM6 (TRPM6 HA ) immunoprecipitated with HA-agarose and probed on WB with antibodies recognizing proteins of interest. Parental 293T cells (WT) served as negative controls. ( B ) Extracts of 293T cells stably expressing FLAG-tagged full-length TRPM6 or M6CK (first amino acid is residue 1365 of full-length TRPM6) immunoprecipitated with FLAG-agarose and probed on Western blot with antibodies recognizing proteins of interest. Parental 293T cells (WT) served as negative controls.
Figure Legend Snippet: M6CK binds PRMT5, WDR77, and ICLN subunits of the protein methylase complex. ( A ) Endogenous TRPM6 binds endogenous PRMT5 and associated proteins. Extracts of 293T cells with endogenous HA-tagged TRPM6 (TRPM6 HA ) immunoprecipitated with HA-agarose and probed on WB with antibodies recognizing proteins of interest. Parental 293T cells (WT) served as negative controls. ( B ) Extracts of 293T cells stably expressing FLAG-tagged full-length TRPM6 or M6CK (first amino acid is residue 1365 of full-length TRPM6) immunoprecipitated with FLAG-agarose and probed on Western blot with antibodies recognizing proteins of interest. Parental 293T cells (WT) served as negative controls.

Techniques Used: Immunoprecipitation, Western Blot, Stable Transfection, Expressing

47) Product Images from "Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo"

Article Title: Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0201880

ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.
Figure Legend Snippet: ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.

Techniques Used: Sequencing, Binding Assay, Mutagenesis, Transfection, Incubation, SDS Page, Staining, Western Blot, Immunoprecipitation, Lysis

Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.
Figure Legend Snippet: Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.

Techniques Used: Infection, Mass Spectrometry, Incubation, SDS Page, Western Blot

48) Product Images from "Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo"

Article Title: Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0201880

ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.
Figure Legend Snippet: ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.

Techniques Used: Sequencing, Binding Assay, Mutagenesis, Transfection, Incubation, SDS Page, Staining, Western Blot, Immunoprecipitation, Lysis

Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.
Figure Legend Snippet: Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.

Techniques Used: Infection, Mass Spectrometry, Incubation, SDS Page, Western Blot

49) Product Images from "A secreted soluble form of ApoE receptor 2 acts as a dominant-negative receptor and inhibits Reelin signaling"

Article Title: A secreted soluble form of ApoE receptor 2 acts as a dominant-negative receptor and inhibits Reelin signaling

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf599

Fig. 5. Analysis of splice variants of ApoER2 and detection of soluble ApoER2 in primary mouse neuronal cultures. ( A ) mRNA from embryonic brain from wild-type mice (lane 1), reeler mice (lane 3) and primary neuronal cultures (E15–16) (lane 2) was used for cDNA synthesis with reverse transcriptase and the resulting cDNA was used for PCR amplification as described in Materials and methods. Amplified products were separated on a 1.5% agarose gel. ( B ) Embryonic brains (E15–16) were dissected and the cerebrum (lane 1), the cerebellum (lane 2) and the olfactory bulbs (lane 3) were used for cDNA synthesis and PCR was performed as in (A). ( C ) Supernatant from a primary neuronal culture (E15–16) was immunoprecipitated with anti-Reelin antibody (G10). Immunocomplexes were precipitated with a mixture of protein A– and protein G–Sepharose and resolved on a 4.5–18% gradient polyacrylamide gel and transferred to nitrocellulose as described in Materials and methods. The soluble ApoER2 fragment was detected by western blotting using an antibody against the first ligand-binding repeat (220). Binding of the primary antibody was visualized with HRP–goat anti-rabbit (1:10 000) and a chemiluminescence system.
Figure Legend Snippet: Fig. 5. Analysis of splice variants of ApoER2 and detection of soluble ApoER2 in primary mouse neuronal cultures. ( A ) mRNA from embryonic brain from wild-type mice (lane 1), reeler mice (lane 3) and primary neuronal cultures (E15–16) (lane 2) was used for cDNA synthesis with reverse transcriptase and the resulting cDNA was used for PCR amplification as described in Materials and methods. Amplified products were separated on a 1.5% agarose gel. ( B ) Embryonic brains (E15–16) were dissected and the cerebrum (lane 1), the cerebellum (lane 2) and the olfactory bulbs (lane 3) were used for cDNA synthesis and PCR was performed as in (A). ( C ) Supernatant from a primary neuronal culture (E15–16) was immunoprecipitated with anti-Reelin antibody (G10). Immunocomplexes were precipitated with a mixture of protein A– and protein G–Sepharose and resolved on a 4.5–18% gradient polyacrylamide gel and transferred to nitrocellulose as described in Materials and methods. The soluble ApoER2 fragment was detected by western blotting using an antibody against the first ligand-binding repeat (220). Binding of the primary antibody was visualized with HRP–goat anti-rabbit (1:10 000) and a chemiluminescence system.

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Immunoprecipitation, Western Blot, Ligand Binding Assay, Binding Assay

Fig. 1. Western blot analysis of cell extracts and supernatants of 293 cells expressing distinct ApoER2 variants. ( A ) Cartoon of the ApoER2 variants expressed in 293 cells and the recombinant soluble receptor fragment produced in E.coli . Numbered circles represent distinct ligand-binding repeats. F represents the domain carrying the furin cleavage site. A, B and C describe the cysteine-rich repeats of the EGF-homology domain. TM marks the transmembrane domain. MBP, maltose-binding protein; His, His 6 tag. ( B ) 293 cells were transfected with ApoER2Δ4-6,8-F (lane 1), ApoER2Δ4-6 (lane 2) or empty vector (lane 3). Ten micrograms of the corresponding cell extracts were separated on a 10% SDS–PAGE gel under reducing conditions and transferred to a nitrocellulose membrane. ApoER2 was detected by western blotting using an antibody recognizing the first ligand-binding repeat (220). Binding of the primary antibody was visualized with HRP–goat anti-rabbit and a chemiluminescence system. ( C ) As (B), but western blotting was performed using an antibody against the cytoplasmic domain of the receptor (20). ( D ) 293 cells were transfected with ApoER2Δ4-6,8-F (lanes 1 and 4), ApoER2Δ4-6 (lane 2) or empty vector (lanes 3 and 5). The respective cell culture supernatants (1.5 ml) were incubated with 50 µl of RAP–Sepharose beads (lanes 1–3) or directly precipitated with TCA (lanes 4 and 5). The precipitated material was separated on a 15% SDS–PAGE gel under reducing (lanes 1–3) or non-reducing (lanes 4 and 5) conditions. For detection of the receptor fragment, Ab 220 was used in a western blot as described for (B). ( E ) 293 cells were transfected with ApoER2Δ4-6,8-F (lanes 1 and 2), ApoER2Δ4-6 (lanes 3 and 4) or empty vector (lanes 5 and 6) and incubated in the presence (+) or absence (–) of the furin inhibitor decanoyl-RVKR-chloromethylketone. The respective cell culture supernatants were used for detection of the soluble receptor fragment as described for (D).
Figure Legend Snippet: Fig. 1. Western blot analysis of cell extracts and supernatants of 293 cells expressing distinct ApoER2 variants. ( A ) Cartoon of the ApoER2 variants expressed in 293 cells and the recombinant soluble receptor fragment produced in E.coli . Numbered circles represent distinct ligand-binding repeats. F represents the domain carrying the furin cleavage site. A, B and C describe the cysteine-rich repeats of the EGF-homology domain. TM marks the transmembrane domain. MBP, maltose-binding protein; His, His 6 tag. ( B ) 293 cells were transfected with ApoER2Δ4-6,8-F (lane 1), ApoER2Δ4-6 (lane 2) or empty vector (lane 3). Ten micrograms of the corresponding cell extracts were separated on a 10% SDS–PAGE gel under reducing conditions and transferred to a nitrocellulose membrane. ApoER2 was detected by western blotting using an antibody recognizing the first ligand-binding repeat (220). Binding of the primary antibody was visualized with HRP–goat anti-rabbit and a chemiluminescence system. ( C ) As (B), but western blotting was performed using an antibody against the cytoplasmic domain of the receptor (20). ( D ) 293 cells were transfected with ApoER2Δ4-6,8-F (lanes 1 and 4), ApoER2Δ4-6 (lane 2) or empty vector (lanes 3 and 5). The respective cell culture supernatants (1.5 ml) were incubated with 50 µl of RAP–Sepharose beads (lanes 1–3) or directly precipitated with TCA (lanes 4 and 5). The precipitated material was separated on a 15% SDS–PAGE gel under reducing (lanes 1–3) or non-reducing (lanes 4 and 5) conditions. For detection of the receptor fragment, Ab 220 was used in a western blot as described for (B). ( E ) 293 cells were transfected with ApoER2Δ4-6,8-F (lanes 1 and 2), ApoER2Δ4-6 (lanes 3 and 4) or empty vector (lanes 5 and 6) and incubated in the presence (+) or absence (–) of the furin inhibitor decanoyl-RVKR-chloromethylketone. The respective cell culture supernatants were used for detection of the soluble receptor fragment as described for (D).

Techniques Used: Western Blot, Expressing, Recombinant, Produced, Ligand Binding Assay, Binding Assay, Transfection, Plasmid Preparation, SDS Page, Cell Culture, Incubation

50) Product Images from "Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo"

Article Title: Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0201880

ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.
Figure Legend Snippet: ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.

Techniques Used: Sequencing, Binding Assay, Mutagenesis, Transfection, Incubation, SDS Page, Staining, Western Blot, Immunoprecipitation, Lysis

Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.
Figure Legend Snippet: Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.

Techniques Used: Infection, Mass Spectrometry, Incubation, SDS Page, Western Blot

51) Product Images from "Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9"

Article Title: Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025694

Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.
Figure Legend Snippet: Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.

Techniques Used: Silver Staining, Binding Assay, Incubation, Immunoprecipitation, Western Blot, Construct, Staining, Marker

Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.
Figure Legend Snippet: Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.

Techniques Used: Sequencing, Affinity Purification, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Incubation, Recombinant

52) Product Images from "Regulation of the MDM2-p53 pathway by the nucleolar protein CSIG in response to nucleolar stress"

Article Title: Regulation of the MDM2-p53 pathway by the nucleolar protein CSIG in response to nucleolar stress

Journal: Scientific Reports

doi: 10.1038/srep36171

CSIG inhibits MDM2-mediated ubiquitination of p53. ( a ) H1299 cells were transfected with the indicated plasmids. 24 h after transfection, the cells were treated with 20 μM MG132 for 6 h. Then, the cells were lysed and subjected to western blot analysis with the indicated antibodies. The asterisk indicates the specific MDM2 band. ( b ) H1299 cells were transfected with the indicated plasmids for 24 h. The cells were incubated with 20 μM MG132 for 6 h before harvesting, after which the cells were lysed under denaturing conditions and incubated with Ni-NTA Agarose (QIAGEN). Cell lysates and Ni-NTA Agarose-bound proteins were analyzed by western blotting with the indicated antibodies. The asterisk indicates the specific MDM2 band. ( c ) SDS-PAGE analysis and Coomassie blue staining of the purified components used for in vitro ubiquitination experiments. ( d ) For in vitro ubiquitination experiments, purified FLAG-p53 was incubated at 37 °C for 1 h with E1, E2, Ub, GST-MDM2, and increasing amounts of purified FLAG-HA-CSIG as indicated, and then analyzed by western blotting. ( e ) Purified FLAG-p53 was mixed with E1, E2, Ub and GST-MDM2 in the absence (lanes 1–4) or presence (lanes 5–8) of FLAG-HA-CSIG for in vitro ubiquitination reactions. The mixture was incubated at 37 °C for the indicated amount of time, and then the reactions were analyzed by western blotting.
Figure Legend Snippet: CSIG inhibits MDM2-mediated ubiquitination of p53. ( a ) H1299 cells were transfected with the indicated plasmids. 24 h after transfection, the cells were treated with 20 μM MG132 for 6 h. Then, the cells were lysed and subjected to western blot analysis with the indicated antibodies. The asterisk indicates the specific MDM2 band. ( b ) H1299 cells were transfected with the indicated plasmids for 24 h. The cells were incubated with 20 μM MG132 for 6 h before harvesting, after which the cells were lysed under denaturing conditions and incubated with Ni-NTA Agarose (QIAGEN). Cell lysates and Ni-NTA Agarose-bound proteins were analyzed by western blotting with the indicated antibodies. The asterisk indicates the specific MDM2 band. ( c ) SDS-PAGE analysis and Coomassie blue staining of the purified components used for in vitro ubiquitination experiments. ( d ) For in vitro ubiquitination experiments, purified FLAG-p53 was incubated at 37 °C for 1 h with E1, E2, Ub, GST-MDM2, and increasing amounts of purified FLAG-HA-CSIG as indicated, and then analyzed by western blotting. ( e ) Purified FLAG-p53 was mixed with E1, E2, Ub and GST-MDM2 in the absence (lanes 1–4) or presence (lanes 5–8) of FLAG-HA-CSIG for in vitro ubiquitination reactions. The mixture was incubated at 37 °C for the indicated amount of time, and then the reactions were analyzed by western blotting.

Techniques Used: Transfection, Western Blot, Incubation, SDS Page, Staining, Purification, In Vitro

CSIG interacts with MDM2. ( a ) HEK293T or H1299 cells were co-transfected with pCMV-MDM2 and pIRES-FLAG-HA-CSIG for 48 h. The cell lysates were subjected to IP using the indicated antibodies and then analyzed by western blotting. ( b ) Purified GST or GST-MDM2 proteins were incubated separately with FLAG-HA-CSIG. The bound proteins were separated using Glutathione Sepharose and were detected by western blotting. ( c ) Schematic representation of the full-length CSIG protein (amino acids 1–490) and CSIG truncation mutants (CSIG-NT: amino acids 1–275, which includes the ribosomal L1 domain; CSIG-CT: amino acids 261–490, which includes the lysine-rich domain). HEK293T cells were co-transfected with pCMV-MDM2 and either pIRES-FLAG-HA-CSIG, pIRES-FLAG-HA-CSIG-NT, or pIRES-FLAG-HA-CSIG-CT for 48 h, after which the cell lysates were subjected to IP with the indicated antibodies and then analyzed by western blotting. ( d ) Schematic representation of the full-length MDM2 protein (amino acids 1–489) and MDM2 truncation mutants (MDM2-D1: amino acids 1–147, which includes the p53 binding domain; MDM2-D2: amino acids 148–348, which includes the central acidic domain; MDM2-D3: amino acids 349–489, which includes the RING finger domain). Purified GST, GST-MDM2-D1, GST-MDM2-D2, or GST-MDM2-D3 proteins were incubated separately with FLAG-HA-CSIG. The bound proteins were separated using Glutathione Sepharose and were analyzed by western blotting. ( e ) U2OS cells were treated with 5 nM ActD for 6 h, then with 20 μM MG132 for an additional 6 h. The cells were then harvested and subjected to IP using the indicated antibodies and then analyzed by western blotting. ( f ) Purified GST or GST-MDM2 proteins were incubated separately with FLAG-p53 and an increasing amount of FLAG-HA-CSIG as indicated. The bound proteins were separated using Glutathione Sepharose and were analyzed by western blotting.
Figure Legend Snippet: CSIG interacts with MDM2. ( a ) HEK293T or H1299 cells were co-transfected with pCMV-MDM2 and pIRES-FLAG-HA-CSIG for 48 h. The cell lysates were subjected to IP using the indicated antibodies and then analyzed by western blotting. ( b ) Purified GST or GST-MDM2 proteins were incubated separately with FLAG-HA-CSIG. The bound proteins were separated using Glutathione Sepharose and were detected by western blotting. ( c ) Schematic representation of the full-length CSIG protein (amino acids 1–490) and CSIG truncation mutants (CSIG-NT: amino acids 1–275, which includes the ribosomal L1 domain; CSIG-CT: amino acids 261–490, which includes the lysine-rich domain). HEK293T cells were co-transfected with pCMV-MDM2 and either pIRES-FLAG-HA-CSIG, pIRES-FLAG-HA-CSIG-NT, or pIRES-FLAG-HA-CSIG-CT for 48 h, after which the cell lysates were subjected to IP with the indicated antibodies and then analyzed by western blotting. ( d ) Schematic representation of the full-length MDM2 protein (amino acids 1–489) and MDM2 truncation mutants (MDM2-D1: amino acids 1–147, which includes the p53 binding domain; MDM2-D2: amino acids 148–348, which includes the central acidic domain; MDM2-D3: amino acids 349–489, which includes the RING finger domain). Purified GST, GST-MDM2-D1, GST-MDM2-D2, or GST-MDM2-D3 proteins were incubated separately with FLAG-HA-CSIG. The bound proteins were separated using Glutathione Sepharose and were analyzed by western blotting. ( e ) U2OS cells were treated with 5 nM ActD for 6 h, then with 20 μM MG132 for an additional 6 h. The cells were then harvested and subjected to IP using the indicated antibodies and then analyzed by western blotting. ( f ) Purified GST or GST-MDM2 proteins were incubated separately with FLAG-p53 and an increasing amount of FLAG-HA-CSIG as indicated. The bound proteins were separated using Glutathione Sepharose and were analyzed by western blotting.

Techniques Used: Transfection, Western Blot, Purification, Incubation, Binding Assay

53) Product Images from "Bora Downregulation Results in Radioresistance by Promoting Repair of Double Strand Breaks"

Article Title: Bora Downregulation Results in Radioresistance by Promoting Repair of Double Strand Breaks

Journal: PLoS ONE

doi: 10.1371/journal.pone.0119208

Bora inhibits MDC1 foci formation via interaction with MDC1 BRCT domain in a phosphorylation-dependent manner. A. Bora interacts with MDC1. IP was performed using anti-Bora antibody followed by blotting with anti-MDC1 antibody in 293T cells. B. Bora interacts with MDC1 via the MDC1 BRCT domain. Lysates from 293T cells overexpressing FLAG-tagged Bora were incubated with GST-BRCT or GST-FHA fusion protein immobilized on the glutathione agarose beads for 2 h before washing. The elution was subsequently analyzed by Western blot with anti-FLAG antibody. C. Effect of phosphorylation on Bora-MDC1 interaction. Lysates from 293T cells overexpressing FLAG-tagged Bora and HA-tagged MDC1 was either incubated with buffer alone or with lambda phosphatase for 15 min at 30°C. The mixture was then incubated with FLAG beads. There was a significant decrease in the binding between Bora and HA-tagged MDC1 in the presence of lambda phosphatase regardless of IR treatment. D. Bora C terminus fragment (313–559 aa), but not N terminus (1–312 aa) co-immunoprecipitates with HA-tagged MDC1. 293T cells were co-transfected with plasmids encoding FLAG-tagged Bora or various deletion constructs, and plasmids encoding HA-tagged MDC1. Lysates were incubated with FLAG beads, followed by Western blot analysis with anti-HA antibody.
Figure Legend Snippet: Bora inhibits MDC1 foci formation via interaction with MDC1 BRCT domain in a phosphorylation-dependent manner. A. Bora interacts with MDC1. IP was performed using anti-Bora antibody followed by blotting with anti-MDC1 antibody in 293T cells. B. Bora interacts with MDC1 via the MDC1 BRCT domain. Lysates from 293T cells overexpressing FLAG-tagged Bora were incubated with GST-BRCT or GST-FHA fusion protein immobilized on the glutathione agarose beads for 2 h before washing. The elution was subsequently analyzed by Western blot with anti-FLAG antibody. C. Effect of phosphorylation on Bora-MDC1 interaction. Lysates from 293T cells overexpressing FLAG-tagged Bora and HA-tagged MDC1 was either incubated with buffer alone or with lambda phosphatase for 15 min at 30°C. The mixture was then incubated with FLAG beads. There was a significant decrease in the binding between Bora and HA-tagged MDC1 in the presence of lambda phosphatase regardless of IR treatment. D. Bora C terminus fragment (313–559 aa), but not N terminus (1–312 aa) co-immunoprecipitates with HA-tagged MDC1. 293T cells were co-transfected with plasmids encoding FLAG-tagged Bora or various deletion constructs, and plasmids encoding HA-tagged MDC1. Lysates were incubated with FLAG beads, followed by Western blot analysis with anti-HA antibody.

Techniques Used: Incubation, Western Blot, Binding Assay, Transfection, Construct

Bora S325A phosphorylation mutant causes increased MDC1 and 53BP1 IRIF formation, DNA repair and resistance to IR treatment. A. Bora S325 phosphorylation is required for its association with MDC1. Lysates from 293T cells overexpressing FLAG-tagged WT Bora, the S325E mutant and the S325A mutant with or without IR treatment were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads and subsequent analysis by Western blotting with anti-FLAG antibody. B. Effect of Bora deletion and mutant constructs on MDC1 and 53BP1 IRIF formation. Left Panel: Foci formation. Hela cells were transfected with wild type, Bora deletion, S501A or S325A mutant FLAG-tagged constructs. Forty-eight h after the transfection, cells were treated with 10 Gy IR and immunostained with indicated antibodies. Right Panel: Quantification of MDC1 and 53BP1-IRIF formation per cell is shown after 10 Gy IR. Error bars represent SEM calculated based on100 cells. C. Percentage of GFP positive cells observed in DR-GFP reporter assay in Hela cells that overexpressed different Bora mutant or deletion constructs. Data are presented as mean ± SEM from three independent experiments. Significance was calculated between WT Bora and S325A mutant. D. Effect of Bora deletion and S501mutant constructs on the Bora binding to MDC1 and IR sensitivity. Left Panel: Immunoprecipitation. Lysates from 293T cells overexpressing FLAG-tagged WT Bora and Bora mutants as well as Bora N or C terminal constructs were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads, with subsequent Western blot analysis with anti-FLAG antibody. Right Panel: Colony forming assays. Bora stably knockdown cell lines were transfected with WT Bora, S501A or C and N terminal constructs, and then treated with increasing dose of IR and cytotoxicity was determined by colony forming assays. E. Effect of S325 mutant construct on IR sensitivity. HupT3 and Hela cell lines with Bora stably knockdown were transfected with WT Bora and S325A mutant, and then treated with increasing dose of IR and cytotoxicity was determined by MTS assays and colony forming assays. P-values were calculated for the difference in AUC values between WT and S325A mutant.
Figure Legend Snippet: Bora S325A phosphorylation mutant causes increased MDC1 and 53BP1 IRIF formation, DNA repair and resistance to IR treatment. A. Bora S325 phosphorylation is required for its association with MDC1. Lysates from 293T cells overexpressing FLAG-tagged WT Bora, the S325E mutant and the S325A mutant with or without IR treatment were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads and subsequent analysis by Western blotting with anti-FLAG antibody. B. Effect of Bora deletion and mutant constructs on MDC1 and 53BP1 IRIF formation. Left Panel: Foci formation. Hela cells were transfected with wild type, Bora deletion, S501A or S325A mutant FLAG-tagged constructs. Forty-eight h after the transfection, cells were treated with 10 Gy IR and immunostained with indicated antibodies. Right Panel: Quantification of MDC1 and 53BP1-IRIF formation per cell is shown after 10 Gy IR. Error bars represent SEM calculated based on100 cells. C. Percentage of GFP positive cells observed in DR-GFP reporter assay in Hela cells that overexpressed different Bora mutant or deletion constructs. Data are presented as mean ± SEM from three independent experiments. Significance was calculated between WT Bora and S325A mutant. D. Effect of Bora deletion and S501mutant constructs on the Bora binding to MDC1 and IR sensitivity. Left Panel: Immunoprecipitation. Lysates from 293T cells overexpressing FLAG-tagged WT Bora and Bora mutants as well as Bora N or C terminal constructs were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads, with subsequent Western blot analysis with anti-FLAG antibody. Right Panel: Colony forming assays. Bora stably knockdown cell lines were transfected with WT Bora, S501A or C and N terminal constructs, and then treated with increasing dose of IR and cytotoxicity was determined by colony forming assays. E. Effect of S325 mutant construct on IR sensitivity. HupT3 and Hela cell lines with Bora stably knockdown were transfected with WT Bora and S325A mutant, and then treated with increasing dose of IR and cytotoxicity was determined by MTS assays and colony forming assays. P-values were calculated for the difference in AUC values between WT and S325A mutant.

Techniques Used: Mutagenesis, Incubation, Western Blot, Construct, Transfection, Reporter Assay, Binding Assay, Immunoprecipitation, Stable Transfection

54) Product Images from "Differential Role of ?1C and ?1A Integrin Cytoplasmic Variants in Modulating Focal Adhesion Kinase, Protein Kinase B/AKT, and Ras/Mitogen-activated Protein Kinase Pathways"

Article Title: Differential Role of ?1C and ?1A Integrin Cytoplasmic Variants in Modulating Focal Adhesion Kinase, Protein Kinase B/AKT, and Ras/Mitogen-activated Protein Kinase Pathways

Journal: Molecular Biology of the Cell

doi:

β 1C associates with α 5 , α V , and α 2 subunits. β 1C or β 1A CHO stable cell lines were cultured for 72 h in the absence of tetracycline and surface-labeled with iodine, and exogenous β 1 integrins were immunoprecipitated with P4C10 (lanes 5 and 10). The immunoprecipitated material was then eluted from protein A–Sepharose with 10 mM Tris-HCl, pH 7.5, 0.5% SDS for 10 min at 70°C, reprecipitated with rabbit antiserum to α 4 (lanes 1 and 6), α 2 (lanes 2 and 7), α V (lanes 3 and 8), or α 5 (lanes 4 and 9), and separated by 10% SDS-PAGE. Lanes 1–5, β 1C CHO; lanes 6–10, β 1A CHO. Proteins were detected by autoradiography. Prestained marker proteins (in kilodaltons) are shown.
Figure Legend Snippet: β 1C associates with α 5 , α V , and α 2 subunits. β 1C or β 1A CHO stable cell lines were cultured for 72 h in the absence of tetracycline and surface-labeled with iodine, and exogenous β 1 integrins were immunoprecipitated with P4C10 (lanes 5 and 10). The immunoprecipitated material was then eluted from protein A–Sepharose with 10 mM Tris-HCl, pH 7.5, 0.5% SDS for 10 min at 70°C, reprecipitated with rabbit antiserum to α 4 (lanes 1 and 6), α 2 (lanes 2 and 7), α V (lanes 3 and 8), or α 5 (lanes 4 and 9), and separated by 10% SDS-PAGE. Lanes 1–5, β 1C CHO; lanes 6–10, β 1A CHO. Proteins were detected by autoradiography. Prestained marker proteins (in kilodaltons) are shown.

Techniques Used: Stable Transfection, Cell Culture, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Marker

Surface expression of β 1C and β 1A in CHO cells. (A–D) β 1C or β 1A CHO stable cell lines were cultured for 48 h either in the absence (A and B) or in the presence (C and D) of 1 μg/ml tetracycline and analyzed by FACS with TS2/16 mAb to human β 1 integrin, 7E2 mAb to hamster β 1 integrin, or 12CA5 as a negative control, followed by FITC goat anti-mouse immunoglobulin G. Fluorescence intensity is expressed in arbitrary units. FACS analysis of a representative clone for each β 1 variant is shown. Thick gray line, TS2/16; dotted line, 7E2; thin black line, 12CA5. (E) CHO stable cell lines were cultured as in A and B and surface-labeled with Na 125 I; exogenous β 1 integrins were immunoprecipitated with P4C10 mAb to human β 1 integrin (lanes 2 and 4). The immunoprecipitated material was then eluted from protein A–Sepharose with 50 mM Tris-HCl, pH 7.5, 2% SDS and boiled for 5 min. The immunocomplexes were then reprecipitated with rabbit antiserum to the β 1C cytoplasmic domain (lane 6) and separated on 7.5% SDS-PAGE. mAb 1C10 (lanes 1 and 3) or normal rabbit serum (lane 5) were used as negative controls. Lanes 1, 2, 5, and 6, β 1C CHO; lanes 3 and 4, β 1A CHO. Proteins were visualized by autoradiography. Prestained marker proteins (in kilodaltons) are shown.
Figure Legend Snippet: Surface expression of β 1C and β 1A in CHO cells. (A–D) β 1C or β 1A CHO stable cell lines were cultured for 48 h either in the absence (A and B) or in the presence (C and D) of 1 μg/ml tetracycline and analyzed by FACS with TS2/16 mAb to human β 1 integrin, 7E2 mAb to hamster β 1 integrin, or 12CA5 as a negative control, followed by FITC goat anti-mouse immunoglobulin G. Fluorescence intensity is expressed in arbitrary units. FACS analysis of a representative clone for each β 1 variant is shown. Thick gray line, TS2/16; dotted line, 7E2; thin black line, 12CA5. (E) CHO stable cell lines were cultured as in A and B and surface-labeled with Na 125 I; exogenous β 1 integrins were immunoprecipitated with P4C10 mAb to human β 1 integrin (lanes 2 and 4). The immunoprecipitated material was then eluted from protein A–Sepharose with 50 mM Tris-HCl, pH 7.5, 2% SDS and boiled for 5 min. The immunocomplexes were then reprecipitated with rabbit antiserum to the β 1C cytoplasmic domain (lane 6) and separated on 7.5% SDS-PAGE. mAb 1C10 (lanes 1 and 3) or normal rabbit serum (lane 5) were used as negative controls. Lanes 1, 2, 5, and 6, β 1C CHO; lanes 3 and 4, β 1A CHO. Proteins were visualized by autoradiography. Prestained marker proteins (in kilodaltons) are shown.

Techniques Used: Expressing, Stable Transfection, Cell Culture, FACS, Negative Control, Fluorescence, Variant Assay, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Marker

55) Product Images from "Reconstitution and Characterization of Budding Yeast ?-Tubulin Complex"

Article Title: Reconstitution and Characterization of Budding Yeast ?-Tubulin Complex

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.02-01-0607

Yield of Spc110p-GST purified from insect cells is increased by the coexpression of yeast calmodulin, Cmd1p. Western blot with anti-GST shows expression of Spc110p-GST in the starting cleared lysates (S), in the insoluble pellets (P), and collected in the eluates after glutathione-Sepharose beads (E). Loading was equal in S and P samples and 10-fold more in E. Quantitation of the intensity of Spc110p-GST bands shows that fivefold more Spc110p-GST is isolated (E) in the presence of calmodulin.
Figure Legend Snippet: Yield of Spc110p-GST purified from insect cells is increased by the coexpression of yeast calmodulin, Cmd1p. Western blot with anti-GST shows expression of Spc110p-GST in the starting cleared lysates (S), in the insoluble pellets (P), and collected in the eluates after glutathione-Sepharose beads (E). Loading was equal in S and P samples and 10-fold more in E. Quantitation of the intensity of Spc110p-GST bands shows that fivefold more Spc110p-GST is isolated (E) in the presence of calmodulin.

Techniques Used: Purification, Western Blot, Expressing, Quantitation Assay, Isolation

Spc110p/Cmd1p can interact with Spc42p or Spc29p independently of each other. (A) GST-Spc110p/Cmd1p was coexpressed in insect cells with either Spc42p-2PY or Spc29p-FLAG or both. S is the starting cleared lysate and E is the eluate from glutathione-Sepharose beads. Protein samples were analyzed by Western blot with anti-GST, anti-PY, and anti-FLAG antibodies. The lower migrating band detected by anti-GST is a GST-Spc110p breakdown. (B) Interactions at the C terminus of Spc110p with Spc42p or Spc29p do not seem to affect its ability to interact with the Tub4p complex. Insect cells were infected with different combinations of viruses as indicated, and the Tub4p complex was tested for its binding to GST-Spc110p/Cmd1p. Note that the Tub4p complex is present at a 10-fold molar excess to GST-Spc110p/Cmd1p in each cleared crude lysate (our unpublished data). Eluates were analyzed by Western blot.
Figure Legend Snippet: Spc110p/Cmd1p can interact with Spc42p or Spc29p independently of each other. (A) GST-Spc110p/Cmd1p was coexpressed in insect cells with either Spc42p-2PY or Spc29p-FLAG or both. S is the starting cleared lysate and E is the eluate from glutathione-Sepharose beads. Protein samples were analyzed by Western blot with anti-GST, anti-PY, and anti-FLAG antibodies. The lower migrating band detected by anti-GST is a GST-Spc110p breakdown. (B) Interactions at the C terminus of Spc110p with Spc42p or Spc29p do not seem to affect its ability to interact with the Tub4p complex. Insect cells were infected with different combinations of viruses as indicated, and the Tub4p complex was tested for its binding to GST-Spc110p/Cmd1p. Note that the Tub4p complex is present at a 10-fold molar excess to GST-Spc110p/Cmd1p in each cleared crude lysate (our unpublished data). Eluates were analyzed by Western blot.

Techniques Used: Western Blot, Infection, Binding Assay

Spc98p binds most robustly to the N-terminal domain of Spc110p. Insect cells were infected with different combinations of viruses as indicated. (A) Production of soluble Spc97p and Spc98p is greatly enhanced upon coexpression with wild-type Tub4p. Western blots show the cleared lysates derived from insect cells expressing Spc98p or Spc98p and Spc97p with and without the coexpression of Tub4p or Tub4–34p. (B) Tub4p interacts poorly with GST or GST-Spc110p 1–220 . Tub4p was tested for its ability to bind glutathione-Sepharose beads, GST, or GST-Spc110p 1–220 . The Western blot shows the starting cleared lysates (S) (0.25% of total) and the eluates (E) (5% of total) from the glutathione-Sepharose beads. (C) An Spc98p/Tub4p complex binds GST-Spc110p 1–220 as efficiently as an Spc98p/Tub4p/Spc97p complex. Herein, we loaded 0.5% of S and 10% of E. Antibodies used in all Westerns include anti-Spc97p, anti-Spc98p, anti-Tub4p, and anti-GST.
Figure Legend Snippet: Spc98p binds most robustly to the N-terminal domain of Spc110p. Insect cells were infected with different combinations of viruses as indicated. (A) Production of soluble Spc97p and Spc98p is greatly enhanced upon coexpression with wild-type Tub4p. Western blots show the cleared lysates derived from insect cells expressing Spc98p or Spc98p and Spc97p with and without the coexpression of Tub4p or Tub4–34p. (B) Tub4p interacts poorly with GST or GST-Spc110p 1–220 . Tub4p was tested for its ability to bind glutathione-Sepharose beads, GST, or GST-Spc110p 1–220 . The Western blot shows the starting cleared lysates (S) (0.25% of total) and the eluates (E) (5% of total) from the glutathione-Sepharose beads. (C) An Spc98p/Tub4p complex binds GST-Spc110p 1–220 as efficiently as an Spc98p/Tub4p/Spc97p complex. Herein, we loaded 0.5% of S and 10% of E. Antibodies used in all Westerns include anti-Spc97p, anti-Spc98p, anti-Tub4p, and anti-GST.

Techniques Used: Infection, Western Blot, Derivative Assay, Expressing

56) Product Images from "Mechanical Stress Activates Smad Pathway through PKC? to Enhance Interleukin-11 Gene Transcription in Osteoblasts"

Article Title: Mechanical Stress Activates Smad Pathway through PKC? to Enhance Interleukin-11 Gene Transcription in Osteoblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013090

Mechanical stress-activated PKCδ phosphorylates BR-Smads, and phosphorylated BR-Smads interact with ΔFosB/JunD on IL-11 gene promoter. (A) GST-Smad1 fusion protein was mixed with Glutathione-Sepharose and incubated at 4C° for 1 h. The Sepharose beads were then washed and used for GST pull-down assay. mPOBs were lysed in lysis buffer and aliquots of the lysate were then incubated under constant agitation for 1 h at 4C° with GST-Smad1 fusion protein coupled to Glutathione-Sepharose. Complexes were then washed three times and bound proteins were eluted and separated on SDS-PAGE. PKCδ bound to GST-Smad1 fusion protein was detected by Western blot analysis using an anti-PKCδ antibody. (B) Wild-type or Y311F mutant PKCδ were transiently transfected into mPOBs, and cells were exposed to FSS for 30 min. Phosphorylation of exogenous Smad1 was analyzed by Western blot analysis using an anti-phospho Smad1 antibody (upper panel). Proteins from 4 wells were analyzed in each lane, and the experiments were repeated for 3 times with similar results. Results from a representative experiment were presented. In the lower panel, the amount of exogenous phospho-Smad1 was quantitated and expressed as a percentage of the amount of phospho-Smad1 in mPOBs transfected with an empty vector without FSS. The data were means ± S.E.M. for three experiments, and difference between FSS(−) and FSS(+) in each group was analyzed by Student's t test. *p
Figure Legend Snippet: Mechanical stress-activated PKCδ phosphorylates BR-Smads, and phosphorylated BR-Smads interact with ΔFosB/JunD on IL-11 gene promoter. (A) GST-Smad1 fusion protein was mixed with Glutathione-Sepharose and incubated at 4C° for 1 h. The Sepharose beads were then washed and used for GST pull-down assay. mPOBs were lysed in lysis buffer and aliquots of the lysate were then incubated under constant agitation for 1 h at 4C° with GST-Smad1 fusion protein coupled to Glutathione-Sepharose. Complexes were then washed three times and bound proteins were eluted and separated on SDS-PAGE. PKCδ bound to GST-Smad1 fusion protein was detected by Western blot analysis using an anti-PKCδ antibody. (B) Wild-type or Y311F mutant PKCδ were transiently transfected into mPOBs, and cells were exposed to FSS for 30 min. Phosphorylation of exogenous Smad1 was analyzed by Western blot analysis using an anti-phospho Smad1 antibody (upper panel). Proteins from 4 wells were analyzed in each lane, and the experiments were repeated for 3 times with similar results. Results from a representative experiment were presented. In the lower panel, the amount of exogenous phospho-Smad1 was quantitated and expressed as a percentage of the amount of phospho-Smad1 in mPOBs transfected with an empty vector without FSS. The data were means ± S.E.M. for three experiments, and difference between FSS(−) and FSS(+) in each group was analyzed by Student's t test. *p

Techniques Used: Incubation, Pull Down Assay, Lysis, SDS Page, Western Blot, Mutagenesis, Transfection, Plasmid Preparation

BR-Smads form complex with ΔFosB/JunD heterodimer by binding to JunD. (A) V5-tagged ΔFosB, Flag-tagged JunD and Myc-tagged Smad1 were co-transfected into mPOBs. Nuclear lysates were prepared and analyzed for binding of nuclear proteins to DNA probe 1 or 3 by DNA precipitation assay followed by Western blotting using antibodies against V5, Flag and Myc. Nuclear lysates from 4 wells were analyzed in each lane, and the experiments were repeated for 3 times with similar results. Results from a representative experiment were presented. (B) GST-Smad1 fusion protein was bound to Glutathione-Sepharose beads, and Flag-JunD or V5-ΔFosB was incubated with GST-Smad1 fusion protein coupled to Glutathione-Sepharose. Complexes were then washed and bound proteins were eluted and separated on SDS-PAGE. JunD or ΔFosB bound to GST-Smad1 fusion protein was detected by Western blot analysis using an anti-Flag or an anti-V5 antibody. (C) GST-Smad1 fusion protein was bound to Glutathione-Sepharose beads, and Flag-tagged WT or C-terminal truncated JunD was incubated with GST-Smad1 fusion protein coupled to Glutathione-Sepharose. Complexes were then washed and bound proteins were eluted and separated on SDS-PAGE. Flag-tagged WT or C-terminal truncated JunD bound to GST-Smad1 fusion protein was detected by Western blot analysis using an anti-Flag antibody (upper panel). Both JunD (WT) and JunD (1–344) (Δ1) had Smad-binding domain, but JunD (1–321) (Δ2) lacked C-terminal Smad-binding domain (lower panel). (D) V5-tagged ΔFosB and Flag-tagged JunD (WT), JunD (Δ1), or JunD (Δ2) along with Myc-tagged Smad1 were co-transfected into mPOBs. Nuclear lysates were prepared and analyzed for binding of nuclear proteins to DNA probe 1 or 3 by DNA precipitation assay, followed by Western blotting using antibodies against V5, Flag and Myc. Nuclear lysates from 4 wells were analyzed in each lane, and the experiments were repeated for 3 times with similar results. Results from a representative experiment were presented.
Figure Legend Snippet: BR-Smads form complex with ΔFosB/JunD heterodimer by binding to JunD. (A) V5-tagged ΔFosB, Flag-tagged JunD and Myc-tagged Smad1 were co-transfected into mPOBs. Nuclear lysates were prepared and analyzed for binding of nuclear proteins to DNA probe 1 or 3 by DNA precipitation assay followed by Western blotting using antibodies against V5, Flag and Myc. Nuclear lysates from 4 wells were analyzed in each lane, and the experiments were repeated for 3 times with similar results. Results from a representative experiment were presented. (B) GST-Smad1 fusion protein was bound to Glutathione-Sepharose beads, and Flag-JunD or V5-ΔFosB was incubated with GST-Smad1 fusion protein coupled to Glutathione-Sepharose. Complexes were then washed and bound proteins were eluted and separated on SDS-PAGE. JunD or ΔFosB bound to GST-Smad1 fusion protein was detected by Western blot analysis using an anti-Flag or an anti-V5 antibody. (C) GST-Smad1 fusion protein was bound to Glutathione-Sepharose beads, and Flag-tagged WT or C-terminal truncated JunD was incubated with GST-Smad1 fusion protein coupled to Glutathione-Sepharose. Complexes were then washed and bound proteins were eluted and separated on SDS-PAGE. Flag-tagged WT or C-terminal truncated JunD bound to GST-Smad1 fusion protein was detected by Western blot analysis using an anti-Flag antibody (upper panel). Both JunD (WT) and JunD (1–344) (Δ1) had Smad-binding domain, but JunD (1–321) (Δ2) lacked C-terminal Smad-binding domain (lower panel). (D) V5-tagged ΔFosB and Flag-tagged JunD (WT), JunD (Δ1), or JunD (Δ2) along with Myc-tagged Smad1 were co-transfected into mPOBs. Nuclear lysates were prepared and analyzed for binding of nuclear proteins to DNA probe 1 or 3 by DNA precipitation assay, followed by Western blotting using antibodies against V5, Flag and Myc. Nuclear lysates from 4 wells were analyzed in each lane, and the experiments were repeated for 3 times with similar results. Results from a representative experiment were presented.

Techniques Used: Binding Assay, Transfection, Western Blot, Incubation, SDS Page

57) Product Images from "Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect"

Article Title: Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-15-31

ASPL interacts with the p97 N-domain. (a) Schematic diagram of the ASPL domain organization and the various truncations used in the precipitation experiments. (b) Purified 6His-tagged p97 was incubated with GST or the indicated GST-tagged ASPL truncations and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to the 6His-tag on p97 (upper panel). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (c) Schematic diagram of the p97 domain organization and the various truncations used in the precipitation experiments. (d) Purified 6His-tagged p97 and p97 truncations were incubated with GST and GST-tagged ASPL before precipitation and analysis by SDS-PAGE and blotting using antibodies specific for the 6His-tagged p97 proteins (upper panel). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel).
Figure Legend Snippet: ASPL interacts with the p97 N-domain. (a) Schematic diagram of the ASPL domain organization and the various truncations used in the precipitation experiments. (b) Purified 6His-tagged p97 was incubated with GST or the indicated GST-tagged ASPL truncations and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to the 6His-tag on p97 (upper panel). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (c) Schematic diagram of the p97 domain organization and the various truncations used in the precipitation experiments. (d) Purified 6His-tagged p97 and p97 truncations were incubated with GST and GST-tagged ASPL before precipitation and analysis by SDS-PAGE and blotting using antibodies specific for the 6His-tagged p97 proteins (upper panel). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel).

Techniques Used: Purification, Incubation, SDS Page, Staining

ASPL interacts with p97 via the UBX domain. (a) Yeast two-hybrid analyses of p97 using the HIS3 reporter gene. Co-transformation of p97 bait with the indicated p97 binding partner preys supported cell growth under conditions selecting for interaction (in the absence of histidine and the presence of 25 mM 3-aminotriazol (3AT)) (right panel). An empty prey vector served as a negative control. (b) Yeast two-hybrid analyses of ASPL using the HIS3 reporter gene. Co-transformation of ASPL bait with p97 or NSF preys supported cell growth under conditions selecting for interaction (in the absence of histidine and the presence of 25 mM 3-aminotriazol (3AT)) (right panel). An empty prey vector served as a negative control. (c) Purified 6His-tagged p97 was incubated with GST or GST-tagged ASPL and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to p97 (upper panel) Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (d) Purified 6His-tagged NSF was incubated with GST or GST-tagged ASPL and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to the 6His-tag on NSF (upper panels). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). Interaction to NSF was only evident when no detergents were included in the buffer system. In the presence of 0.5% Triton X-100 no interaction between ASPL and NSF was observed. (e) MelJuSo cell lysates were used in immunoprecipitation (IP) experiments with antibodies to ASPL and Protein A Sepharose or as a control Protein A Sepharose beads only. SDS-PAGE and blotting revealed that ASPL co-precipitated p97, but not NSF or the Rpn1 or α subunits of the 26S proteasome.
Figure Legend Snippet: ASPL interacts with p97 via the UBX domain. (a) Yeast two-hybrid analyses of p97 using the HIS3 reporter gene. Co-transformation of p97 bait with the indicated p97 binding partner preys supported cell growth under conditions selecting for interaction (in the absence of histidine and the presence of 25 mM 3-aminotriazol (3AT)) (right panel). An empty prey vector served as a negative control. (b) Yeast two-hybrid analyses of ASPL using the HIS3 reporter gene. Co-transformation of ASPL bait with p97 or NSF preys supported cell growth under conditions selecting for interaction (in the absence of histidine and the presence of 25 mM 3-aminotriazol (3AT)) (right panel). An empty prey vector served as a negative control. (c) Purified 6His-tagged p97 was incubated with GST or GST-tagged ASPL and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to p97 (upper panel) Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (d) Purified 6His-tagged NSF was incubated with GST or GST-tagged ASPL and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to the 6His-tag on NSF (upper panels). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). Interaction to NSF was only evident when no detergents were included in the buffer system. In the presence of 0.5% Triton X-100 no interaction between ASPL and NSF was observed. (e) MelJuSo cell lysates were used in immunoprecipitation (IP) experiments with antibodies to ASPL and Protein A Sepharose or as a control Protein A Sepharose beads only. SDS-PAGE and blotting revealed that ASPL co-precipitated p97, but not NSF or the Rpn1 or α subunits of the 26S proteasome.

Techniques Used: Transformation Assay, Binding Assay, Plasmid Preparation, Negative Control, Purification, Incubation, SDS Page, Staining, Immunoprecipitation

58) Product Images from "RAD54 N-terminal domain is a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions"

Article Title: RAD54 N-terminal domain is a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions

Journal: Nature Communications

doi: 10.1038/s41467-017-02497-x

Effect of CDK2 phosphorylation on the RAD54 activities. a The kinetics of BM of PX junction (no. 71/169/170/171; 10 nM) promoted by RAD54 (60 nM), phosphorylated RAD54 (60 nM), RAD54 S49A (60 nM), or phosphorylated RAD54 S49A (60 nM). b The effect of RAD54 phosphorylation (60 nM) on its ATPase activity was tested using supercoiled pUC19 (2 µM, nt) as DNA substrate. c The experimental scheme of the D-loop reaction. The nucleoprotein filaments were formed between RAD51 and ssDNA for 30 min at 37 °C. The reactions were then moved to 30 °C. Then RAD54 was added followed by addition of pUC19 dsDNA substrate to initiate the reaction. The effect of RAD54 (50 nM) or phosphomimetic RAD54 S49E (50 nM) on D-loop formation by RAD51 (1.25 µM) between 32 P-labeled ssDNA (no. 90; 2.4 µM nts) and pUC19 (50 µM, nts). The D-loops were analyzed by electrophoresis in 1% agarose gel. d Data from c are plotted as a graph. Each experiment was repeated three times. Error bars represent the s.e.m.
Figure Legend Snippet: Effect of CDK2 phosphorylation on the RAD54 activities. a The kinetics of BM of PX junction (no. 71/169/170/171; 10 nM) promoted by RAD54 (60 nM), phosphorylated RAD54 (60 nM), RAD54 S49A (60 nM), or phosphorylated RAD54 S49A (60 nM). b The effect of RAD54 phosphorylation (60 nM) on its ATPase activity was tested using supercoiled pUC19 (2 µM, nt) as DNA substrate. c The experimental scheme of the D-loop reaction. The nucleoprotein filaments were formed between RAD51 and ssDNA for 30 min at 37 °C. The reactions were then moved to 30 °C. Then RAD54 was added followed by addition of pUC19 dsDNA substrate to initiate the reaction. The effect of RAD54 (50 nM) or phosphomimetic RAD54 S49E (50 nM) on D-loop formation by RAD51 (1.25 µM) between 32 P-labeled ssDNA (no. 90; 2.4 µM nts) and pUC19 (50 µM, nts). The D-loops were analyzed by electrophoresis in 1% agarose gel. d Data from c are plotted as a graph. Each experiment was repeated three times. Error bars represent the s.e.m.

Techniques Used: Activity Assay, Labeling, Electrophoresis, Agarose Gel Electrophoresis

The 95 a.a. N-terminal truncation enhances DNA translocation activity of RAD54. a The experimental scheme of the triple-helix displacement assay. The triple-helix forming oligonucleotide (TFO) is paired to the linearized pMJ5 plasmid. The asterisk indicates the 32 P-label. b The kinetics of triple-helix (0.5 nM, molecules) displacement by RAD54 (5 nM) or RAD54 96–747 (5 nM) was analyzed by electrophoresis in 1.2 % agarose gels. c Data from b are plotted as a graph. The values obtained in protein-free reaction (Supplementary Figure 1b ) were subtracted from the values of RAD54-promoted reactions. Each experiment was repeated three times. Error bars represent the s.e.m.
Figure Legend Snippet: The 95 a.a. N-terminal truncation enhances DNA translocation activity of RAD54. a The experimental scheme of the triple-helix displacement assay. The triple-helix forming oligonucleotide (TFO) is paired to the linearized pMJ5 plasmid. The asterisk indicates the 32 P-label. b The kinetics of triple-helix (0.5 nM, molecules) displacement by RAD54 (5 nM) or RAD54 96–747 (5 nM) was analyzed by electrophoresis in 1.2 % agarose gels. c Data from b are plotted as a graph. The values obtained in protein-free reaction (Supplementary Figure 1b ) were subtracted from the values of RAD54-promoted reactions. Each experiment was repeated three times. Error bars represent the s.e.m.

Techniques Used: Translocation Assay, Activity Assay, Plasmid Preparation, Electrophoresis

59) Product Images from "Transcription factor TAFII250 promotes Mdm2-dependent turnover of p53"

Article Title: Transcription factor TAFII250 promotes Mdm2-dependent turnover of p53

Journal: Oncogene

doi: 10.1038/sj.onc.1210209

TAFII250 promotes ubiquitylation and degradation of p53. ( a ) H1299 cells were cotransfected with 1 μ g of p53, 5 μ g of Mdm2 and 5 and 10 μ g of TAFII250 expression vectors. Cells were harvested 36 h after transfection and lysed with 2 × SDS sample buffer. The cells extracts were analysed for p53 protein levels. ( b ) Ni 2+ pull-down was performed using extracts of H1299 cells that had been transfected with 1 μ g of p53, 1 μ g of Mdm2, 1 μ g of TAFII250 and 2 μ g of His-Ubiquitin expression vectors. The His-tagged ubiquitylated proteins were purified with Ni 2+ agarose beads and analysed by western blotting using anti-p53 antibody DO-1. ( c ) p53 ubiquitylation was measured in H1299 cells transfected with plasmids encoding p53, TAFII250 and Mdm2 (wild-type and ΔAD mutant) as indicated. ( d ) Mdm2 (wild-type and ΔAD mutant) auto-ubiquitylation was measured in H1299 cells transfected with plasmids encoding wild type or ΔAD mutant Mdm2.
Figure Legend Snippet: TAFII250 promotes ubiquitylation and degradation of p53. ( a ) H1299 cells were cotransfected with 1 μ g of p53, 5 μ g of Mdm2 and 5 and 10 μ g of TAFII250 expression vectors. Cells were harvested 36 h after transfection and lysed with 2 × SDS sample buffer. The cells extracts were analysed for p53 protein levels. ( b ) Ni 2+ pull-down was performed using extracts of H1299 cells that had been transfected with 1 μ g of p53, 1 μ g of Mdm2, 1 μ g of TAFII250 and 2 μ g of His-Ubiquitin expression vectors. The His-tagged ubiquitylated proteins were purified with Ni 2+ agarose beads and analysed by western blotting using anti-p53 antibody DO-1. ( c ) p53 ubiquitylation was measured in H1299 cells transfected with plasmids encoding p53, TAFII250 and Mdm2 (wild-type and ΔAD mutant) as indicated. ( d ) Mdm2 (wild-type and ΔAD mutant) auto-ubiquitylation was measured in H1299 cells transfected with plasmids encoding wild type or ΔAD mutant Mdm2.

Techniques Used: Expressing, Transfection, Purification, Western Blot, Mutagenesis

TAFII250 promotes the interaction of Mdm2 and p53. ( a ) H1299 cells were transfected with plasmids expressing Mdm2 alone or Mdm2 together with TAFII250. Cell extracts were prepared and the volumes adjusted empirically to yield equal concentrations of Mdm2 in each extract. H1299 cells were also transfected, separately, with a plasmid expressing wild-type human p53. Subsequently, extracts were prepared and equal volumes mixed with the extracts from the Mdm2- or Mdm2/TAFII250-expressing cells. Mdm2 was subsequently immunoprecipitated and the amount of co-immunoprecipitating p53 was measured by Western blotting using the anti-p53 polyclonal antibody, CM-1. ( b ) H1299 cells were transfected with plasmids encoding TAFII250, Mdm2 and wild type or a F19A mutant of p53. Cells were treated with the proteasome inhibitor, MG132, 6 h before harvesting. Mdm2 and p53 were detected by Western blotting following immunoprecipitation of Mdm2 (upper panels). The total levels of Mdm2 and p53 are also shown (lower panels). ( c and d ) GST-Mdm2, GST-Mdm2-ΔAD ( d ) or GST alone were bound to glutathione-sepharose beads and subsequently incubated with p53 WT or a F19A mutant of p53 in the presence or absence of TAFII250. Immobilized proteins were detected by Western blotting.
Figure Legend Snippet: TAFII250 promotes the interaction of Mdm2 and p53. ( a ) H1299 cells were transfected with plasmids expressing Mdm2 alone or Mdm2 together with TAFII250. Cell extracts were prepared and the volumes adjusted empirically to yield equal concentrations of Mdm2 in each extract. H1299 cells were also transfected, separately, with a plasmid expressing wild-type human p53. Subsequently, extracts were prepared and equal volumes mixed with the extracts from the Mdm2- or Mdm2/TAFII250-expressing cells. Mdm2 was subsequently immunoprecipitated and the amount of co-immunoprecipitating p53 was measured by Western blotting using the anti-p53 polyclonal antibody, CM-1. ( b ) H1299 cells were transfected with plasmids encoding TAFII250, Mdm2 and wild type or a F19A mutant of p53. Cells were treated with the proteasome inhibitor, MG132, 6 h before harvesting. Mdm2 and p53 were detected by Western blotting following immunoprecipitation of Mdm2 (upper panels). The total levels of Mdm2 and p53 are also shown (lower panels). ( c and d ) GST-Mdm2, GST-Mdm2-ΔAD ( d ) or GST alone were bound to glutathione-sepharose beads and subsequently incubated with p53 WT or a F19A mutant of p53 in the presence or absence of TAFII250. Immobilized proteins were detected by Western blotting.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis, Incubation

60) Product Images from "RAD54 N-terminal domain is a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions"

Article Title: RAD54 N-terminal domain is a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions

Journal: Nature Communications

doi: 10.1038/s41467-017-02497-x

Effect of CDK2 phosphorylation on the RAD54 activities. a The kinetics of BM of PX junction (no. 71/169/170/171; 10 nM) promoted by RAD54 (60 nM), phosphorylated RAD54 (60 nM), RAD54 S49A (60 nM), or phosphorylated RAD54 S49A (60 nM). b The effect of RAD54 phosphorylation (60 nM) on its ATPase activity was tested using supercoiled pUC19 (2 µM, nt) as DNA substrate. c The experimental scheme of the D-loop reaction. The nucleoprotein filaments were formed between RAD51 and ssDNA for 30 min at 37 °C. The reactions were then moved to 30 °C. Then RAD54 was added followed by addition of pUC19 dsDNA substrate to initiate the reaction. The effect of RAD54 (50 nM) or phosphomimetic RAD54 S49E (50 nM) on D-loop formation by RAD51 (1.25 µM) between 32 P-labeled ssDNA (no. 90; 2.4 µM nts) and pUC19 (50 µM, nts). The D-loops were analyzed by electrophoresis in 1% agarose gel. d Data from c are plotted as a graph. Each experiment was repeated three times. Error bars represent the s.e.m.
Figure Legend Snippet: Effect of CDK2 phosphorylation on the RAD54 activities. a The kinetics of BM of PX junction (no. 71/169/170/171; 10 nM) promoted by RAD54 (60 nM), phosphorylated RAD54 (60 nM), RAD54 S49A (60 nM), or phosphorylated RAD54 S49A (60 nM). b The effect of RAD54 phosphorylation (60 nM) on its ATPase activity was tested using supercoiled pUC19 (2 µM, nt) as DNA substrate. c The experimental scheme of the D-loop reaction. The nucleoprotein filaments were formed between RAD51 and ssDNA for 30 min at 37 °C. The reactions were then moved to 30 °C. Then RAD54 was added followed by addition of pUC19 dsDNA substrate to initiate the reaction. The effect of RAD54 (50 nM) or phosphomimetic RAD54 S49E (50 nM) on D-loop formation by RAD51 (1.25 µM) between 32 P-labeled ssDNA (no. 90; 2.4 µM nts) and pUC19 (50 µM, nts). The D-loops were analyzed by electrophoresis in 1% agarose gel. d Data from c are plotted as a graph. Each experiment was repeated three times. Error bars represent the s.e.m.

Techniques Used: Activity Assay, Labeling, Electrophoresis, Agarose Gel Electrophoresis

The 95 a.a. N-terminal truncation enhances DNA translocation activity of RAD54. a The experimental scheme of the triple-helix displacement assay. The triple-helix forming oligonucleotide (TFO) is paired to the linearized pMJ5 plasmid. The asterisk indicates the 32 P-label. b The kinetics of triple-helix (0.5 nM, molecules) displacement by RAD54 (5 nM) or RAD54 96–747 (5 nM) was analyzed by electrophoresis in 1.2 % agarose gels. c Data from b ) were subtracted from the values of RAD54-promoted reactions. Each experiment was repeated three times. Error bars represent the s.e.m.
Figure Legend Snippet: The 95 a.a. N-terminal truncation enhances DNA translocation activity of RAD54. a The experimental scheme of the triple-helix displacement assay. The triple-helix forming oligonucleotide (TFO) is paired to the linearized pMJ5 plasmid. The asterisk indicates the 32 P-label. b The kinetics of triple-helix (0.5 nM, molecules) displacement by RAD54 (5 nM) or RAD54 96–747 (5 nM) was analyzed by electrophoresis in 1.2 % agarose gels. c Data from b ) were subtracted from the values of RAD54-promoted reactions. Each experiment was repeated three times. Error bars represent the s.e.m.

Techniques Used: Translocation Assay, Activity Assay, Plasmid Preparation, Electrophoresis

61) Product Images from "Arabidopsis D-Type Cyclin CYCD4;1 Is a Novel Cyclin Partner of B2-Type Cyclin-Dependent Kinase 1"

Article Title: Arabidopsis D-Type Cyclin CYCD4;1 Is a Novel Cyclin Partner of B2-Type Cyclin-Dependent Kinase 1

Journal: Plant Physiology

doi: 10.1104/pp.103.020644

Arabidopsis cyclins interacting with CDKB2;1. In vitro pull-down assay was conducted with D-type and mitotic cyclins. A, [ 35 S]Met-labeled CYCD1;1, CYCD2;1, CYCD3;1, and CYCD4;1 were incubated with GST (lane 2) or GST-CDKB2;1 (lane 3) immobilized on glutathione Sepharose beads. Proteins bound to the beads were separated by SDS-PAGE and autoradiographed. One-microliter input of the translation products is included as a control (lane 1). Arrowheads indicate the position of in vitro translated cyclins. B, CYCA2;2 and CYCB2;1 were incubated with the beads as described above.
Figure Legend Snippet: Arabidopsis cyclins interacting with CDKB2;1. In vitro pull-down assay was conducted with D-type and mitotic cyclins. A, [ 35 S]Met-labeled CYCD1;1, CYCD2;1, CYCD3;1, and CYCD4;1 were incubated with GST (lane 2) or GST-CDKB2;1 (lane 3) immobilized on glutathione Sepharose beads. Proteins bound to the beads were separated by SDS-PAGE and autoradiographed. One-microliter input of the translation products is included as a control (lane 1). Arrowheads indicate the position of in vitro translated cyclins. B, CYCA2;2 and CYCB2;1 were incubated with the beads as described above.

Techniques Used: In Vitro, Pull Down Assay, Labeling, Incubation, SDS Page

Arabidopsis CDKs interacting with CYCD1;1 or CYCD4;1. In vitro pull-down assay was conducted with CDKAs and CDKBs. [ 35 S]Met-labeled CYCD1;1 or CYCD4;1 was incubated with GST (lane 2), GST-CDKA;1 (lane 3), GST-CDKB1;1 (lane 4), or GST-CDKB2:1 (lane 5) immobilized on glutathione Sepharose beads. Proteins bound to the beads were separated by SDS-PAGE and autoradiographed. One microliter of in vitro-translated products is included as a control (lane 1).
Figure Legend Snippet: Arabidopsis CDKs interacting with CYCD1;1 or CYCD4;1. In vitro pull-down assay was conducted with CDKAs and CDKBs. [ 35 S]Met-labeled CYCD1;1 or CYCD4;1 was incubated with GST (lane 2), GST-CDKA;1 (lane 3), GST-CDKB1;1 (lane 4), or GST-CDKB2:1 (lane 5) immobilized on glutathione Sepharose beads. Proteins bound to the beads were separated by SDS-PAGE and autoradiographed. One microliter of in vitro-translated products is included as a control (lane 1).

Techniques Used: In Vitro, Pull Down Assay, Labeling, Incubation, SDS Page

62) Product Images from "Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor"

Article Title: Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.80

CLSP is transported across the blood-CSF barrier into the CSF. ( a ) Purified hCLSP-MycHis, which was generated in E. coli , was i.p. injected into mice. For i.p.1, 100 μ l of the sample containing 1.7 nmol of hCLSP, was injected into a mouse three times with an interval of 1 h. For i.p.2, 300 μ l of the sample containing 5.1 nmol of hCLSP was injected at once. For i.n. administration, 0.34 nmol of hCLSP-MycHis in 20 μ l of PBS was i.n. administered at once. At 1 h from the last administration of hCLSP-MycHis, CSFs were collected from the i.p.1, i.p.2 and i.n. mice and 10 μ l of CSF was analyzed by SDS-PAGE and immunoblot analysis using the antibody against hCLSP (top). The indicated amounts of serum, collected from i.p.2 mouse, were similarly analyzed (bottom). Indicated femtomolar amounts of recombinant hCLSP were similarly processed for the estimation of the hCLSP amounts. The possible contamination of blood to CSF during the collection of CSF was estimated to be far less than 1%. ( b ) An experiment similar to that in the panel ( a ) was performed using another mouse. At 1 h affter i.p. injection of 5.0 nmol of hCLSP-MycHis, serum and CSF were collected. 0.1 μ l of serum and 10 μ l of CSF were analyzed with SDS-PAGE and immunoblot analysis with antibodies to hCLSP (top) and mouse IgG (bottom). ( c ) Blood plasma samples (100 μ l) from three normal middle-aged males were subjected to the immunoprecipitation with CLSP antibody (anti-hCLSP)- or preimmune (Pre) serum-conjugated protein G-sepharose 4B. The immunoprecipitates were then fractionated by SDS-PAGE and immunobloted with CLSP antibody. Indicated amounts of recombinant hCLSP-MycHis, produced in bacteria, was simultaneously analyzed for the estimation of the hCLSP amounts in the plasma
Figure Legend Snippet: CLSP is transported across the blood-CSF barrier into the CSF. ( a ) Purified hCLSP-MycHis, which was generated in E. coli , was i.p. injected into mice. For i.p.1, 100 μ l of the sample containing 1.7 nmol of hCLSP, was injected into a mouse three times with an interval of 1 h. For i.p.2, 300 μ l of the sample containing 5.1 nmol of hCLSP was injected at once. For i.n. administration, 0.34 nmol of hCLSP-MycHis in 20 μ l of PBS was i.n. administered at once. At 1 h from the last administration of hCLSP-MycHis, CSFs were collected from the i.p.1, i.p.2 and i.n. mice and 10 μ l of CSF was analyzed by SDS-PAGE and immunoblot analysis using the antibody against hCLSP (top). The indicated amounts of serum, collected from i.p.2 mouse, were similarly analyzed (bottom). Indicated femtomolar amounts of recombinant hCLSP were similarly processed for the estimation of the hCLSP amounts. The possible contamination of blood to CSF during the collection of CSF was estimated to be far less than 1%. ( b ) An experiment similar to that in the panel ( a ) was performed using another mouse. At 1 h affter i.p. injection of 5.0 nmol of hCLSP-MycHis, serum and CSF were collected. 0.1 μ l of serum and 10 μ l of CSF were analyzed with SDS-PAGE and immunoblot analysis with antibodies to hCLSP (top) and mouse IgG (bottom). ( c ) Blood plasma samples (100 μ l) from three normal middle-aged males were subjected to the immunoprecipitation with CLSP antibody (anti-hCLSP)- or preimmune (Pre) serum-conjugated protein G-sepharose 4B. The immunoprecipitates were then fractionated by SDS-PAGE and immunobloted with CLSP antibody. Indicated amounts of recombinant hCLSP-MycHis, produced in bacteria, was simultaneously analyzed for the estimation of the hCLSP amounts in the plasma

Techniques Used: Purification, Generated, Injection, Mouse Assay, SDS Page, Recombinant, Immunoprecipitation, Produced

Secretion is essential for hCLSP activity. ( a ) SH-SY5Y cells were transfected with the empty pFLAG vector (vec) or the pFLAG vector encoding hCLSP or Humanin (HN) (C-terminally FLAG-tagged), and coincubated with or without (+)-Brefeldin A (BFA) (20 ng/ml) for 6 h. The CM were subjected to the immunoprecipitatation with M2 FLAG-antibody-conjugated agarose. The resulting immunoprecipitates and input cell lysates were subjected to SDS-PAGE and immunoblot analysis with the M2 FLAG antibody. ( b ) The structures of the hCLSP deletion mutants (all C-terminally tagged with MycHis). EHR; endogenous Humanin-like region (see text). ( c – h ) SH-SY5Y cells, co-transfected with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP) together with the pcDNA3.1/MycHis vector (vector) or the pcDNA3.1/MycHis encoding hCLSP or one of hCLSP deletion mutants (all C-terminally tagged with MycHis), were harvested for WST-8 assays ( c , e and g ). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with indicated antibodies ( d , f and h ). CM were also pulled down with Talon metal beads and the precipitates were subjected to SDS-PAGE and immunoblot analysis with indicated antibodies ( d , bottom panel; f , left bottom panel; h , bottom panel). The 6 × His antibody was used only for the detection of hCLSP, hCLSP-ΔC1 and hCLSP-ΔC2 in the CM in ( d ) *** P
Figure Legend Snippet: Secretion is essential for hCLSP activity. ( a ) SH-SY5Y cells were transfected with the empty pFLAG vector (vec) or the pFLAG vector encoding hCLSP or Humanin (HN) (C-terminally FLAG-tagged), and coincubated with or without (+)-Brefeldin A (BFA) (20 ng/ml) for 6 h. The CM were subjected to the immunoprecipitatation with M2 FLAG-antibody-conjugated agarose. The resulting immunoprecipitates and input cell lysates were subjected to SDS-PAGE and immunoblot analysis with the M2 FLAG antibody. ( b ) The structures of the hCLSP deletion mutants (all C-terminally tagged with MycHis). EHR; endogenous Humanin-like region (see text). ( c – h ) SH-SY5Y cells, co-transfected with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP) together with the pcDNA3.1/MycHis vector (vector) or the pcDNA3.1/MycHis encoding hCLSP or one of hCLSP deletion mutants (all C-terminally tagged with MycHis), were harvested for WST-8 assays ( c , e and g ). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with indicated antibodies ( d , f and h ). CM were also pulled down with Talon metal beads and the precipitates were subjected to SDS-PAGE and immunoblot analysis with indicated antibodies ( d , bottom panel; f , left bottom panel; h , bottom panel). The 6 × His antibody was used only for the detection of hCLSP, hCLSP-ΔC1 and hCLSP-ΔC2 in the CM in ( d ) *** P

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, SDS Page

63) Product Images from "Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding *"

Article Title: Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.669002

Binding of C-tail with 9-1-1 and 9 ΔC -1-1. A and B, glutathione-Sepharose beads pre-bound with GST only or GST/FLAG-tagged C-tails (from E. coli cells in A , and EC and HF in B from E. coli and insect cells, respectively) were mixed with 10 pmol each of purified 9-1-1 or 9 ΔC -1-1 at 4 °C for 1 h. After washing the beads, input proteins (1%) and bound proteins (20%) were analyzed by immunoblotting using the indicated antibodies for 9-1-1 subunits ( upper panel ), and staining with Coomassie Brilliant Blue ( A ) or Ponceau S ( B ) for C-tail ( lower panels ). Asterisks indicate degraded C-tail fragments.
Figure Legend Snippet: Binding of C-tail with 9-1-1 and 9 ΔC -1-1. A and B, glutathione-Sepharose beads pre-bound with GST only or GST/FLAG-tagged C-tails (from E. coli cells in A , and EC and HF in B from E. coli and insect cells, respectively) were mixed with 10 pmol each of purified 9-1-1 or 9 ΔC -1-1 at 4 °C for 1 h. After washing the beads, input proteins (1%) and bound proteins (20%) were analyzed by immunoblotting using the indicated antibodies for 9-1-1 subunits ( upper panel ), and staining with Coomassie Brilliant Blue ( A ) or Ponceau S ( B ) for C-tail ( lower panels ). Asterisks indicate degraded C-tail fragments.

Techniques Used: Binding Assay, Purification, Staining

Analyses of amino acid sequence of C-tail necessary for its binding to 9 ΔC -1-1. A, schematic illustration of human C-tail and its deletions used in this study. C-tail, C-N, and C-C contain aa 272–391, aa 272–332, and aa 333–391, respectively. Constructs Δ336, Δ341, Δ351 d5 , Δ351, Δ356, Δ361, and Δ371 harbor internal deletions of aa 336–345, aa 341–350, aa 351–355, aa 351–360, aa 356–365, aa 361–370, and aa 371–380 from C-C, respectively; C-tail Δ351d5 and C-tail Δ351 are the same deletions as Δ351 d5 and Δ351, respectively, from the C-tail. C-tail FFAA harbors Ala substitutions at both Phe-365 and Phe-366, which were expressed as fusions with GST/FLAG tags at their N-terminal portion. Below is the sequence of the C-tail, including the 15-aa stretch (aa 351–375) required for interaction with CRS. The NLS and FF motif (Phe-365/Phe-366) are boxed in white and black , respectively. B, glutathione-Sepharose beads pre-bound with GST only ( mock ), GST/FLAG-tagged C-tail ( C-tail ), C-tail mutant ( C-tail FFAA ), or its deletions ( C-N, C-C, Δ 336, Δ 341, Δ 351 d5 , Δ 351, Δ 356, Δ 361, Δ 371, C-tail Δ 351d5 , and C-tail Δ 351 ) were mixed with 10 pmol alone ( left panel ) or 5 and 10 pmol (+, and ++, respectively) ( right panel ) of purified 9 ΔC -1-1, and incubated at 4 °C for 1 h. After washing the beads, 50 fmol ( left panel ) and 100 fmol ( right panel ) of purified 9 ΔC -1-1 and 10% ( left panel ) or 20% ( right panel ) of the bound fractions were analyzed by immunoblotting using anti-Rad1 antibody ( upper panels ) and staining with Ponceau S for the C-tail and its deletions ( lower panels ). Asterisks indicate degraded C-tail fragments.
Figure Legend Snippet: Analyses of amino acid sequence of C-tail necessary for its binding to 9 ΔC -1-1. A, schematic illustration of human C-tail and its deletions used in this study. C-tail, C-N, and C-C contain aa 272–391, aa 272–332, and aa 333–391, respectively. Constructs Δ336, Δ341, Δ351 d5 , Δ351, Δ356, Δ361, and Δ371 harbor internal deletions of aa 336–345, aa 341–350, aa 351–355, aa 351–360, aa 356–365, aa 361–370, and aa 371–380 from C-C, respectively; C-tail Δ351d5 and C-tail Δ351 are the same deletions as Δ351 d5 and Δ351, respectively, from the C-tail. C-tail FFAA harbors Ala substitutions at both Phe-365 and Phe-366, which were expressed as fusions with GST/FLAG tags at their N-terminal portion. Below is the sequence of the C-tail, including the 15-aa stretch (aa 351–375) required for interaction with CRS. The NLS and FF motif (Phe-365/Phe-366) are boxed in white and black , respectively. B, glutathione-Sepharose beads pre-bound with GST only ( mock ), GST/FLAG-tagged C-tail ( C-tail ), C-tail mutant ( C-tail FFAA ), or its deletions ( C-N, C-C, Δ 336, Δ 341, Δ 351 d5 , Δ 351, Δ 356, Δ 361, Δ 371, C-tail Δ 351d5 , and C-tail Δ 351 ) were mixed with 10 pmol alone ( left panel ) or 5 and 10 pmol (+, and ++, respectively) ( right panel ) of purified 9 ΔC -1-1, and incubated at 4 °C for 1 h. After washing the beads, 50 fmol ( left panel ) and 100 fmol ( right panel ) of purified 9 ΔC -1-1 and 10% ( left panel ) or 20% ( right panel ) of the bound fractions were analyzed by immunoblotting using anti-Rad1 antibody ( upper panels ) and staining with Ponceau S for the C-tail and its deletions ( lower panels ). Asterisks indicate degraded C-tail fragments.

Techniques Used: Sequencing, Binding Assay, Construct, Mutagenesis, Purification, Incubation, Staining

64) Product Images from "Transcription factor Sp3 is silenced through SUMO modification by PIAS1"

Article Title: Transcription factor Sp3 is silenced through SUMO modification by PIAS1

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf510

Fig. 3. Identification of PIAS1 as an interaction partner of Sp3 and Ubc9. ( A ) Interaction of PIAS1 with the ID of Sp3 in Saccharomyces cerevisiae . Yeast cells containing a LexA-driven LacZ reporter construct were transformed with expression constructs for LexA, LexA-Sp3ID or LexA-Sp3ID/kee (baits) along with a construct in which the Gal4 activation domain is fused to the 500 C-terminal amino acids of PIAS1 (Gal4-PIAS1, prey). In the LexA-Sp3ID/kee construct, the KEE sequence of the SUMOylation motif is replaced by three alanine residues. β-galactosidase activity was visualized by addition of 0.5% X-gal to the agar. ( B ) In vitro association of PIAS1 with Sp3 and SUMO-1-modified Sp3. Sp3 (small isoform) was in vitro translated in the presence of [ 35 S]methionine and subsequently subjected to in vitro SUMO-1 conjugation. The reaction that contained unmodified Sp3 and SUMO-modified Sp3 (lane 8) was incubated with similar amounts of the glutathione matrix (lane 2), immobilized GST (lane 3), GST–Ubc9 (lane 4) or GST–PIAS1 (lane 6). In lane 5, unmodified 35 S-labelled Sp3 was incubated with GST–PIAS1. Bound Sp3 proteins were resolved by SDS–PAGE and visualized by fluorography. Lanes 7 and 8 contain 10% of the input 35 S-labelled Sp3 protein. Numbers on the left indicate the molecular mass of protein markers in kDa. ( C ) In vitro association of Ubc9 with PIAS1. PIAS1 was in vitro translated in the presence of [ 35 S]methionine and incubated with glutathione–Sepharose matrix (lane 2) or with ∼2 µg of immobilized GST (lanes 3 and 4) or GST–Ubc9 (lanes 5 and 6). Bound PIAS1 protein was resolved by SDS–PAGE and visualized by fluorography. Lane 7 contains 10% of the input 35 S-labelled PIAS1 protein. Numbers on the left indicate the molecular mass of protein markers in kDa.
Figure Legend Snippet: Fig. 3. Identification of PIAS1 as an interaction partner of Sp3 and Ubc9. ( A ) Interaction of PIAS1 with the ID of Sp3 in Saccharomyces cerevisiae . Yeast cells containing a LexA-driven LacZ reporter construct were transformed with expression constructs for LexA, LexA-Sp3ID or LexA-Sp3ID/kee (baits) along with a construct in which the Gal4 activation domain is fused to the 500 C-terminal amino acids of PIAS1 (Gal4-PIAS1, prey). In the LexA-Sp3ID/kee construct, the KEE sequence of the SUMOylation motif is replaced by three alanine residues. β-galactosidase activity was visualized by addition of 0.5% X-gal to the agar. ( B ) In vitro association of PIAS1 with Sp3 and SUMO-1-modified Sp3. Sp3 (small isoform) was in vitro translated in the presence of [ 35 S]methionine and subsequently subjected to in vitro SUMO-1 conjugation. The reaction that contained unmodified Sp3 and SUMO-modified Sp3 (lane 8) was incubated with similar amounts of the glutathione matrix (lane 2), immobilized GST (lane 3), GST–Ubc9 (lane 4) or GST–PIAS1 (lane 6). In lane 5, unmodified 35 S-labelled Sp3 was incubated with GST–PIAS1. Bound Sp3 proteins were resolved by SDS–PAGE and visualized by fluorography. Lanes 7 and 8 contain 10% of the input 35 S-labelled Sp3 protein. Numbers on the left indicate the molecular mass of protein markers in kDa. ( C ) In vitro association of Ubc9 with PIAS1. PIAS1 was in vitro translated in the presence of [ 35 S]methionine and incubated with glutathione–Sepharose matrix (lane 2) or with ∼2 µg of immobilized GST (lanes 3 and 4) or GST–Ubc9 (lanes 5 and 6). Bound PIAS1 protein was resolved by SDS–PAGE and visualized by fluorography. Lane 7 contains 10% of the input 35 S-labelled PIAS1 protein. Numbers on the left indicate the molecular mass of protein markers in kDa.

Techniques Used: Construct, Transformation Assay, Expressing, Activation Assay, Sequencing, Activity Assay, In Vitro, Modification, Conjugation Assay, Incubation, SDS Page

Fig. 2. In vitro SUMOylation and deSUMOylation of Sp3 fragments. ( A ) Schematic drawing of the conjugation pathway leading to SUMOylation of Sp3. The free carboxyl group of the C-terminal glycine of SUMO forms an isopeptide bond with the ε-amino group of a lysine (K) in Sp3. The reaction is mediated by the ATP-dependent heterodimeric E1 enzyme Aos1/Uba2 and the E2 enzyme Ubc9 that form thioesters (S) with SUMO. ( B ) Affinity-purified epitope-tagged Sp3WT (lanes 1–3) and Sp3SD (lanes 5–7) were subjected to in vitro SUMOylation reactions in the presence or absence of recombinant E1, Ubc9 and SUMO-1 as indicated. Sp3 and SUMO-modified Sp3 (arrow) were detected by western blot analysis using anti-HA antibodies. Lane 4 (HA/FL-Sp3) contains whole-cell extract from Sp3-expressing SL2 cells. ( C ) Bacterially expressed GST fusion proteins GST–Sp3WT, GST–Sp3kee and GST–Sp3BID bound to GST–Sepharose were subjected to in vitro SUMOylation reactions in the presence or absence of recombinant E1, Ubc9 and SUMO-1 as indicated. The GST–Sp3BID protein contains the second glutamine-rich activation domain (B domain) and the ID with the IKEE motif lacking the transactivation domain A and the C-terminal DNA-binding domain of Sp3. In the GST–Sp3kee protein, the KEE wild-type sequence of the ID is replaced by three alanine residues. Reaction products were detected by western blot analysis using anti-Sp3 (αSp3) and anti-SUMO-1 (αSUMO-1) antibodies as indicated. Arrows point to the SUMOylated Sp3 fragments. ( D ) SUMO-1 and SUMO-2 were equally conjugated to Sp3. Epitope-tagged recombinant Sp3 wild-type (Sp3WT) or the Sp3SD mutant was subjected to SUMO modification with equal concentrations of SUMO-1 and SUMO-2 (5 ng/µl each). Detection was by immunoblotting with αHA antibodies. ( E ) DeSUMOylation of SUMO-1-modified Sp3 by the isopeptidase Ulp1. The GST–Sp3BID fragment (see panel C) bound to glutathione–Sepharose was SUMOylated in vitro and subsequently incubated with recombinant ULP1 isopeptidase at 16 or 30°C for 30 or 60 min, as indicated. Detection was by immunoblotting with αSp3 antibodies.
Figure Legend Snippet: Fig. 2. In vitro SUMOylation and deSUMOylation of Sp3 fragments. ( A ) Schematic drawing of the conjugation pathway leading to SUMOylation of Sp3. The free carboxyl group of the C-terminal glycine of SUMO forms an isopeptide bond with the ε-amino group of a lysine (K) in Sp3. The reaction is mediated by the ATP-dependent heterodimeric E1 enzyme Aos1/Uba2 and the E2 enzyme Ubc9 that form thioesters (S) with SUMO. ( B ) Affinity-purified epitope-tagged Sp3WT (lanes 1–3) and Sp3SD (lanes 5–7) were subjected to in vitro SUMOylation reactions in the presence or absence of recombinant E1, Ubc9 and SUMO-1 as indicated. Sp3 and SUMO-modified Sp3 (arrow) were detected by western blot analysis using anti-HA antibodies. Lane 4 (HA/FL-Sp3) contains whole-cell extract from Sp3-expressing SL2 cells. ( C ) Bacterially expressed GST fusion proteins GST–Sp3WT, GST–Sp3kee and GST–Sp3BID bound to GST–Sepharose were subjected to in vitro SUMOylation reactions in the presence or absence of recombinant E1, Ubc9 and SUMO-1 as indicated. The GST–Sp3BID protein contains the second glutamine-rich activation domain (B domain) and the ID with the IKEE motif lacking the transactivation domain A and the C-terminal DNA-binding domain of Sp3. In the GST–Sp3kee protein, the KEE wild-type sequence of the ID is replaced by three alanine residues. Reaction products were detected by western blot analysis using anti-Sp3 (αSp3) and anti-SUMO-1 (αSUMO-1) antibodies as indicated. Arrows point to the SUMOylated Sp3 fragments. ( D ) SUMO-1 and SUMO-2 were equally conjugated to Sp3. Epitope-tagged recombinant Sp3 wild-type (Sp3WT) or the Sp3SD mutant was subjected to SUMO modification with equal concentrations of SUMO-1 and SUMO-2 (5 ng/µl each). Detection was by immunoblotting with αHA antibodies. ( E ) DeSUMOylation of SUMO-1-modified Sp3 by the isopeptidase Ulp1. The GST–Sp3BID fragment (see panel C) bound to glutathione–Sepharose was SUMOylated in vitro and subsequently incubated with recombinant ULP1 isopeptidase at 16 or 30°C for 30 or 60 min, as indicated. Detection was by immunoblotting with αSp3 antibodies.

Techniques Used: In Vitro, Conjugation Assay, Affinity Purification, Recombinant, Modification, Western Blot, Expressing, Activation Assay, Binding Assay, Sequencing, Mutagenesis, Incubation

65) Product Images from "The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex"

Article Title: The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex

Journal: Genes & Development

doi: 10.1101/gad.2038011

U2AF and PRP19C are required for CTD-dependent splicing activity. ( A ) NF20–40 was depleted of U2AF65 at 1 M KCl using Poly-U sepharose beads, followed by immunoblotting against U2AF65 and CDC5L. Band intensities quantified using Li-cor software are indicated below . ( B , lanes 2 and 3 ) Mock and depleted extracts from A were used in the CTD-dependent splicing assay. One-hundred nanomolar U2AF65 and U2AF produced in baculovirus-infected insect cells were tested for their ability to complement the depleted NF20–40 (lanes 4 , 5 ) or activate splicing when added alone (lanes 6 , 7 ). Relative intensities of spliced product were quantified using ImageQuant software and are indicated below . ( C ) PRP19C was depleted from an active Mono Q fraction at 500 mM NaCl using an anti-CDC5L antibody or was mock-depleted using an anti-Flag antibody. Mock and depleted samples were immunoblotted for U2AF65 and CDC5L. Relative CDC5L levels were normalized to U2AF65 and are indicated below . ( D ) CTD-dependent splicing assays were performed using the mock- and PRP19C-depleted samples from C . Relative amounts of spliced product are indicated below .
Figure Legend Snippet: U2AF and PRP19C are required for CTD-dependent splicing activity. ( A ) NF20–40 was depleted of U2AF65 at 1 M KCl using Poly-U sepharose beads, followed by immunoblotting against U2AF65 and CDC5L. Band intensities quantified using Li-cor software are indicated below . ( B , lanes 2 and 3 ) Mock and depleted extracts from A were used in the CTD-dependent splicing assay. One-hundred nanomolar U2AF65 and U2AF produced in baculovirus-infected insect cells were tested for their ability to complement the depleted NF20–40 (lanes 4 , 5 ) or activate splicing when added alone (lanes 6 , 7 ). Relative intensities of spliced product were quantified using ImageQuant software and are indicated below . ( C ) PRP19C was depleted from an active Mono Q fraction at 500 mM NaCl using an anti-CDC5L antibody or was mock-depleted using an anti-Flag antibody. Mock and depleted samples were immunoblotted for U2AF65 and CDC5L. Relative CDC5L levels were normalized to U2AF65 and are indicated below . ( D ) CTD-dependent splicing assays were performed using the mock- and PRP19C-depleted samples from C . Relative amounts of spliced product are indicated below .

Techniques Used: Activity Assay, Software, Splicing Assay, Produced, Infection

U2AF65 interacts with PRP19C in an RNA-independent manner. ( A , left ) Schematic of U2AF65 and deletion constructs used for GST pull-downs. ( Right ) These constructs were expressed in E. coli and then purified by GSH sepharose beads. The beads were run on SDS-PAGE and Coomassie-stained. ( B ) GST pull-down was performed using GST-tagged U2AF65, U2AF65ΔRS, or U2AF65ΔUHM. After a 3-h incubation at 22°C with the indicated extract, beads were washed and then eluted with 15 mM glutathione. Eluted proteins were immunoblotted with an anti-Prp19 antibody. ( C ) A vector expressing Flag-tagged SPF27 was transfected into 293T cells, cells were harvested and used to make NE, and PRP19C was purified using anti-Flag agarose and washed with buffer D containing the indicated salt concentration. In parallel, beads were incubated with NE from nontransfected cells amM KCl. Beads were eluted with Flag peptide, separated by SDS-PAGE, and Coomassie stained ( left ) or immunoblotted with the indicated antibody ( right ). ( D ) Co-IPs were carried out in NF20–40 using anti-Prp19 antibodies or anti-GST antibodies (mock). Beads were washed with buffer D and then boiled and immunoblotted for U2AF65. ( E ) Co-IP was performed using anti-CDC5L antibodies or anti-GST antibodies as above, except NF20–40 was mock- or RNase A-treated beforehand for 30 min at 37°C. The effectiveness of RNase A digestion was confirmed by agarose gel electrophoresis followed by ethidium staining (not shown).
Figure Legend Snippet: U2AF65 interacts with PRP19C in an RNA-independent manner. ( A , left ) Schematic of U2AF65 and deletion constructs used for GST pull-downs. ( Right ) These constructs were expressed in E. coli and then purified by GSH sepharose beads. The beads were run on SDS-PAGE and Coomassie-stained. ( B ) GST pull-down was performed using GST-tagged U2AF65, U2AF65ΔRS, or U2AF65ΔUHM. After a 3-h incubation at 22°C with the indicated extract, beads were washed and then eluted with 15 mM glutathione. Eluted proteins were immunoblotted with an anti-Prp19 antibody. ( C ) A vector expressing Flag-tagged SPF27 was transfected into 293T cells, cells were harvested and used to make NE, and PRP19C was purified using anti-Flag agarose and washed with buffer D containing the indicated salt concentration. In parallel, beads were incubated with NE from nontransfected cells amM KCl. Beads were eluted with Flag peptide, separated by SDS-PAGE, and Coomassie stained ( left ) or immunoblotted with the indicated antibody ( right ). ( D ) Co-IPs were carried out in NF20–40 using anti-Prp19 antibodies or anti-GST antibodies (mock). Beads were washed with buffer D and then boiled and immunoblotted for U2AF65. ( E ) Co-IP was performed using anti-CDC5L antibodies or anti-GST antibodies as above, except NF20–40 was mock- or RNase A-treated beforehand for 30 min at 37°C. The effectiveness of RNase A digestion was confirmed by agarose gel electrophoresis followed by ethidium staining (not shown).

Techniques Used: Construct, Purification, SDS Page, Staining, Incubation, Plasmid Preparation, Expressing, Transfection, Concentration Assay, Co-Immunoprecipitation Assay, Agarose Gel Electrophoresis

66) Product Images from "C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *"

Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M806152200

Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.
Figure Legend Snippet: Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.

Techniques Used: Activation Assay, Purification, Western Blot, Incubation, Activity Assay, Expressing, Mutagenesis, Cell Cycle Assay

Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.
Figure Legend Snippet: Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.

Techniques Used: Activation Assay, Purification, Western Blot, Incubation, Activity Assay

67) Product Images from "Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *"

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.036491

Identification of the LEDGF-Cdc7-ASK interaction. A , schematics showing the domain organization of LEDGF and the cTAP-tagged LEDGF-(326–530) construct. Locations of the PWWP domain, NLS, AT-hooks, and IBD of LEDGF, calmodulin binding peptide ( CBP ), tobacco etch virus ( TEV ) protease site, and the IgG binding module from S. aureus protein A ( protA ) of the cTAP tag are indicated. B , co-IP experiments. HeLa cells were transiently transfected with HA-tagged mouse LEDGF (mLEDGF), human LEDGF, LEDGF-(326–530), HRP2, mouse p52 ( mp52 ), or an empty vector. Whole cell extracts ( WCE ; lanes 1–6 ) or proteins pulled down with anti-HA affinity matrix from whole cell extracts ( lanes 7–12 ) were tested by Western blotting using anti-HA, anti-Cdc7, and anti-β-actin antibodies. Migration positions of protein molecular mass standards (kDa), and the heavy chain of mouse IgG ( IgG H ) are indicated. C , IP of endogenous proteins. Extracts from untransfected 293T cells were incubated with rabbit anti-LEDGF antibody ( lane 3 ) or control rabbit IgG ( lane 4 ) and protein G-agarose, and the recovered proteins were analyzed by Western blotting with anti-Cdc7 and anti-ASK antibodies. Lanes 1 and 2 contained whole cell extract. To improve detection of ASK, the samples in lanes 2–4 were treated with λ-protein phosphatase (λ PPase ). The bands corresponding to ASK are indicated with asterisks .
Figure Legend Snippet: Identification of the LEDGF-Cdc7-ASK interaction. A , schematics showing the domain organization of LEDGF and the cTAP-tagged LEDGF-(326–530) construct. Locations of the PWWP domain, NLS, AT-hooks, and IBD of LEDGF, calmodulin binding peptide ( CBP ), tobacco etch virus ( TEV ) protease site, and the IgG binding module from S. aureus protein A ( protA ) of the cTAP tag are indicated. B , co-IP experiments. HeLa cells were transiently transfected with HA-tagged mouse LEDGF (mLEDGF), human LEDGF, LEDGF-(326–530), HRP2, mouse p52 ( mp52 ), or an empty vector. Whole cell extracts ( WCE ; lanes 1–6 ) or proteins pulled down with anti-HA affinity matrix from whole cell extracts ( lanes 7–12 ) were tested by Western blotting using anti-HA, anti-Cdc7, and anti-β-actin antibodies. Migration positions of protein molecular mass standards (kDa), and the heavy chain of mouse IgG ( IgG H ) are indicated. C , IP of endogenous proteins. Extracts from untransfected 293T cells were incubated with rabbit anti-LEDGF antibody ( lane 3 ) or control rabbit IgG ( lane 4 ) and protein G-agarose, and the recovered proteins were analyzed by Western blotting with anti-Cdc7 and anti-ASK antibodies. Lanes 1 and 2 contained whole cell extract. To improve detection of ASK, the samples in lanes 2–4 were treated with λ-protein phosphatase (λ PPase ). The bands corresponding to ASK are indicated with asterisks .

Techniques Used: Construct, Binding Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Migration, Incubation

Residues 625–674 of ASK are required for binding to LEDGF. A , schematic of ASK truncations. Locations of the N, M, and C motifs are indicated. B , C terminus of ASK is required for the interaction with LEDGF. S-tagged Cdc7-ASK or its indicated mutant forms were incubated with S-protein-agarose in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. Lane 14 contains a mock pull-down of LEDGF with S-protein-agarose. Input quantities of proteins ( lanes 1–5 ) or proteins captured on the beads ( lanes 6–14 ) separated by SDS-PAGE were stained with Coomassie Blue ( top ) and analyzed by Western blotting using anti-LEDGF antibody ( bottom ). C , deletion of 50 residues from the C terminus of ASK is sufficient to ablate the interaction with LEDGF. Cdc7-ASK or its mutants were incubated with S-protein-agarose beads in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. LEDGF was incubated with S-protein-agarose alone in lane 14. Lanes 1–5 contained input levels of the indicated proteins. Samples were analyzed as in B .
Figure Legend Snippet: Residues 625–674 of ASK are required for binding to LEDGF. A , schematic of ASK truncations. Locations of the N, M, and C motifs are indicated. B , C terminus of ASK is required for the interaction with LEDGF. S-tagged Cdc7-ASK or its indicated mutant forms were incubated with S-protein-agarose in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. Lane 14 contains a mock pull-down of LEDGF with S-protein-agarose. Input quantities of proteins ( lanes 1–5 ) or proteins captured on the beads ( lanes 6–14 ) separated by SDS-PAGE were stained with Coomassie Blue ( top ) and analyzed by Western blotting using anti-LEDGF antibody ( bottom ). C , deletion of 50 residues from the C terminus of ASK is sufficient to ablate the interaction with LEDGF. Cdc7-ASK or its mutants were incubated with S-protein-agarose beads in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. LEDGF was incubated with S-protein-agarose alone in lane 14. Lanes 1–5 contained input levels of the indicated proteins. Samples were analyzed as in B .

Techniques Used: Binding Assay, Mutagenesis, Incubation, SDS Page, Staining, Western Blot

68) Product Images from "Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *"

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.036491

Identification of the LEDGF-Cdc7-ASK interaction. A , schematics showing the domain organization of LEDGF and the cTAP-tagged LEDGF-(326–530) construct. Locations of the PWWP domain, NLS, AT-hooks, and IBD of LEDGF, calmodulin binding peptide ( CBP ), tobacco etch virus ( TEV ) protease site, and the IgG binding module from S. aureus protein A ( protA ) of the cTAP tag are indicated. B , co-IP experiments. HeLa cells were transiently transfected with HA-tagged mouse LEDGF (mLEDGF), human LEDGF, LEDGF-(326–530), HRP2, mouse p52 ( mp52 ), or an empty vector. Whole cell extracts ( WCE ; lanes 1–6 ) or proteins pulled down with anti-HA affinity matrix from whole cell extracts ( lanes 7–12 ) were tested by Western blotting using anti-HA, anti-Cdc7, and anti-β-actin antibodies. Migration positions of protein molecular mass standards (kDa), and the heavy chain of mouse IgG ( IgG H ) are indicated. C , IP of endogenous proteins. Extracts from untransfected 293T cells were incubated with rabbit anti-LEDGF antibody ( lane 3 ) or control rabbit IgG ( lane 4 ) and protein G-agarose, and the recovered proteins were analyzed by Western blotting with anti-Cdc7 and anti-ASK antibodies. Lanes 1 and 2 contained whole cell extract. To improve detection of ASK, the samples in lanes 2–4 were treated with λ-protein phosphatase (λ PPase ). The bands corresponding to ASK are indicated with asterisks .
Figure Legend Snippet: Identification of the LEDGF-Cdc7-ASK interaction. A , schematics showing the domain organization of LEDGF and the cTAP-tagged LEDGF-(326–530) construct. Locations of the PWWP domain, NLS, AT-hooks, and IBD of LEDGF, calmodulin binding peptide ( CBP ), tobacco etch virus ( TEV ) protease site, and the IgG binding module from S. aureus protein A ( protA ) of the cTAP tag are indicated. B , co-IP experiments. HeLa cells were transiently transfected with HA-tagged mouse LEDGF (mLEDGF), human LEDGF, LEDGF-(326–530), HRP2, mouse p52 ( mp52 ), or an empty vector. Whole cell extracts ( WCE ; lanes 1–6 ) or proteins pulled down with anti-HA affinity matrix from whole cell extracts ( lanes 7–12 ) were tested by Western blotting using anti-HA, anti-Cdc7, and anti-β-actin antibodies. Migration positions of protein molecular mass standards (kDa), and the heavy chain of mouse IgG ( IgG H ) are indicated. C , IP of endogenous proteins. Extracts from untransfected 293T cells were incubated with rabbit anti-LEDGF antibody ( lane 3 ) or control rabbit IgG ( lane 4 ) and protein G-agarose, and the recovered proteins were analyzed by Western blotting with anti-Cdc7 and anti-ASK antibodies. Lanes 1 and 2 contained whole cell extract. To improve detection of ASK, the samples in lanes 2–4 were treated with λ-protein phosphatase (λ PPase ). The bands corresponding to ASK are indicated with asterisks .

Techniques Used: Construct, Binding Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Migration, Incubation

Residues 625–674 of ASK are required for binding to LEDGF. A , schematic of ASK truncations. Locations of the N, M, and C motifs are indicated. B , C terminus of ASK is required for the interaction with LEDGF. S-tagged Cdc7-ASK or its indicated mutant forms were incubated with S-protein-agarose in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. Lane 14 contains a mock pull-down of LEDGF with S-protein-agarose. Input quantities of proteins ( lanes 1–5 ) or proteins captured on the beads ( lanes 6–14 ) separated by SDS-PAGE were stained with Coomassie Blue ( top ) and analyzed by Western blotting using anti-LEDGF antibody ( bottom ). C , deletion of 50 residues from the C terminus of ASK is sufficient to ablate the interaction with LEDGF. Cdc7-ASK or its mutants were incubated with S-protein-agarose beads in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. LEDGF was incubated with S-protein-agarose alone in lane 14. Lanes 1–5 contained input levels of the indicated proteins. Samples were analyzed as in B .
Figure Legend Snippet: Residues 625–674 of ASK are required for binding to LEDGF. A , schematic of ASK truncations. Locations of the N, M, and C motifs are indicated. B , C terminus of ASK is required for the interaction with LEDGF. S-tagged Cdc7-ASK or its indicated mutant forms were incubated with S-protein-agarose in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. Lane 14 contains a mock pull-down of LEDGF with S-protein-agarose. Input quantities of proteins ( lanes 1–5 ) or proteins captured on the beads ( lanes 6–14 ) separated by SDS-PAGE were stained with Coomassie Blue ( top ) and analyzed by Western blotting using anti-LEDGF antibody ( bottom ). C , deletion of 50 residues from the C terminus of ASK is sufficient to ablate the interaction with LEDGF. Cdc7-ASK or its mutants were incubated with S-protein-agarose beads in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. LEDGF was incubated with S-protein-agarose alone in lane 14. Lanes 1–5 contained input levels of the indicated proteins. Samples were analyzed as in B .

Techniques Used: Binding Assay, Mutagenesis, Incubation, SDS Page, Staining, Western Blot

69) Product Images from "The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA"

Article Title: The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr385

( A ) Organization of the HIV-2 leader RNA. ( B ) Loose dimer formation by wt HIV-2 leader RNA and its UTRh3 mutant, in the absence or presence of antisense oligonucleotide probe1 (P1) (TBM agarose gel). ( C ) Influence of antisense oligonucleotides, probe1 (P1; directed to SL1) and probe2 (P2; directed to TAR hairpin III), on the formation of the loose dimers by +1–560 RNA (TBM agarose gel). ( D ) Tight dimer formation of the +1–560 RNA (TBE PAA gel). For analyses of dimer formation, samples were incubated at 37°C for the times indicated, with some sample incubated for an additional 10 or 15 min (parenthesis) in the presence of P1 and/or P2. Lanes F correspond to the formamide-denatured control sample. RNA monomers are indicated as M, dimers as D. ( E ) NMIA modification of the leader RNA mutant UTRh3. Lane (−) represents control sample with untreated RNA; lane (+) NMIA modification, A, U, C and G are sequencing lanes.
Figure Legend Snippet: ( A ) Organization of the HIV-2 leader RNA. ( B ) Loose dimer formation by wt HIV-2 leader RNA and its UTRh3 mutant, in the absence or presence of antisense oligonucleotide probe1 (P1) (TBM agarose gel). ( C ) Influence of antisense oligonucleotides, probe1 (P1; directed to SL1) and probe2 (P2; directed to TAR hairpin III), on the formation of the loose dimers by +1–560 RNA (TBM agarose gel). ( D ) Tight dimer formation of the +1–560 RNA (TBE PAA gel). For analyses of dimer formation, samples were incubated at 37°C for the times indicated, with some sample incubated for an additional 10 or 15 min (parenthesis) in the presence of P1 and/or P2. Lanes F correspond to the formamide-denatured control sample. RNA monomers are indicated as M, dimers as D. ( E ) NMIA modification of the leader RNA mutant UTRh3. Lane (−) represents control sample with untreated RNA; lane (+) NMIA modification, A, U, C and G are sequencing lanes.

Techniques Used: Mutagenesis, Agarose Gel Electrophoresis, Incubation, Modification, Sequencing

( A ) Analysis of the loose dimer formation by the HIV-2 leader RNA (+1–560) in the SHAPE buffers and folding conditions (TBM agarose gel). ( B ) Secondary-structure model of the HIV-2 leader RNA captured in the loose dimer form. Two kissing-loop interfaces are indicated as black frames. Nucleotide residues accessibilities to NMIA are given in colours and according to scale. ( C ) Processed SHAPE reactivities as a function of nucleotide position. Red and orange bars reflect reactive positions in the RNA. Insert on the right represents the box plot analysis of distinct reactivity distributions for the HIV-2 leader RNA. Box outline middle 50% of dataset; the median is shown with heavy line. Circles indicate extreme values ( 7 ): 1.5 times the interquartile range (boxed). Horizontal lines above and below the box are the largest or smallest non-outlier values.
Figure Legend Snippet: ( A ) Analysis of the loose dimer formation by the HIV-2 leader RNA (+1–560) in the SHAPE buffers and folding conditions (TBM agarose gel). ( B ) Secondary-structure model of the HIV-2 leader RNA captured in the loose dimer form. Two kissing-loop interfaces are indicated as black frames. Nucleotide residues accessibilities to NMIA are given in colours and according to scale. ( C ) Processed SHAPE reactivities as a function of nucleotide position. Red and orange bars reflect reactive positions in the RNA. Insert on the right represents the box plot analysis of distinct reactivity distributions for the HIV-2 leader RNA. Box outline middle 50% of dataset; the median is shown with heavy line. Circles indicate extreme values ( 7 ): 1.5 times the interquartile range (boxed). Horizontal lines above and below the box are the largest or smallest non-outlier values.

Techniques Used: Agarose Gel Electrophoresis

70) Product Images from "ATP and MO25? Regulate the Conformational State of the STRAD? Pseudokinase and Activation of the LKB1 Tumour Suppressor"

Article Title: ATP and MO25? Regulate the Conformational State of the STRAD? Pseudokinase and Activation of the LKB1 Tumour Suppressor

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1000126

Mutation of MO25α concave surface residues abolishes STRADα and LKB1 binding. (A and B) The indicated constructs of GST-STRADα and Myc-MO25α were expressed in 293 cells. Cells 36 h post-transfection were lysed, and GST-STRADα was purified with glutathione-Sepharose. The purified GST-STRADα preparation (upper panels), as well as the cell extracts (lower panels), was immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments. Dotted line indicates the junction of two gels. AL, activation loop. (C and D) Two hundred ninety-three cells were cotransfected with the indicated constructs of GST-LKB1, Flag-STRADα, and Myc-MO25α. Cells 36 h post-transfection were lysed, and GST-LKB1 was purified and assayed for its ability to phosphorylate the LKBtide peptide. Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the standard deviation for one experiment). Affinity-purified GST-LKB1 preparation (upper panel), as well as cell extracts (lower panel), was immunoblotted with the indicated antibodies.
Figure Legend Snippet: Mutation of MO25α concave surface residues abolishes STRADα and LKB1 binding. (A and B) The indicated constructs of GST-STRADα and Myc-MO25α were expressed in 293 cells. Cells 36 h post-transfection were lysed, and GST-STRADα was purified with glutathione-Sepharose. The purified GST-STRADα preparation (upper panels), as well as the cell extracts (lower panels), was immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments. Dotted line indicates the junction of two gels. AL, activation loop. (C and D) Two hundred ninety-three cells were cotransfected with the indicated constructs of GST-LKB1, Flag-STRADα, and Myc-MO25α. Cells 36 h post-transfection were lysed, and GST-LKB1 was purified and assayed for its ability to phosphorylate the LKBtide peptide. Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the standard deviation for one experiment). Affinity-purified GST-LKB1 preparation (upper panel), as well as cell extracts (lower panel), was immunoblotted with the indicated antibodies.

Techniques Used: Mutagenesis, Binding Assay, Construct, Transfection, Purification, Activation Assay, Standard Deviation, Affinity Purification

Interaction of ATP and MO25α with STRADα controls LKB1 activity. (A) The structure of the ATP binding site of STRADα in which the key interacting residues are emphasized. (B) Fluorescence emission spectra (excitation 410 nm) of TNP-ATP (5 µM) bound to wild-type and mutant forms of STRADα (2 µM) and/or wild-type MO25α (2 µM). A reference cuvette containing only TNP-ATP (5 µM) was subtracted as background. A Coomassie Blue-stained SDS-PAGE gel of each form of STRADα analysed is shown (GGK = G76D+G78D+K197E, GGR = G76D+G78D+R215E, and GGKR = G76D+G78D+K197E+R215E). (C) Wild-type GST-LKB1 and indicated forms of Flag-STRADα were expressed in 293 cells in the absence of MO25α. Cells at 36 h posttransfection were lysed and GST-LKB1 affinity purified on glutathione-Sepharose. The purified GST-LKB1 preparation (upper panel), as well as the cell extracts (lower panel), was immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments. Dotted line indicates where the gel was cut. (D and E) 293 cells were co-transfected with the indicated constructs of GST-LKB1, Flag-STRADα, and Myc-MO25α. Cells at 36 h posttransfection were lysed, and GST-LKB1 was affinity purified and assayed for the ability to activate the heterotrimeric AMPK complex expressed in E. coli , as described in Materials and Methods . Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the SD for a single triplicate experiment). Affinity-purified GST-LKB1 preparation (upper panel), as well as cell extracts (lower panel), was immunoblotted with the indicated antibodies.
Figure Legend Snippet: Interaction of ATP and MO25α with STRADα controls LKB1 activity. (A) The structure of the ATP binding site of STRADα in which the key interacting residues are emphasized. (B) Fluorescence emission spectra (excitation 410 nm) of TNP-ATP (5 µM) bound to wild-type and mutant forms of STRADα (2 µM) and/or wild-type MO25α (2 µM). A reference cuvette containing only TNP-ATP (5 µM) was subtracted as background. A Coomassie Blue-stained SDS-PAGE gel of each form of STRADα analysed is shown (GGK = G76D+G78D+K197E, GGR = G76D+G78D+R215E, and GGKR = G76D+G78D+K197E+R215E). (C) Wild-type GST-LKB1 and indicated forms of Flag-STRADα were expressed in 293 cells in the absence of MO25α. Cells at 36 h posttransfection were lysed and GST-LKB1 affinity purified on glutathione-Sepharose. The purified GST-LKB1 preparation (upper panel), as well as the cell extracts (lower panel), was immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments. Dotted line indicates where the gel was cut. (D and E) 293 cells were co-transfected with the indicated constructs of GST-LKB1, Flag-STRADα, and Myc-MO25α. Cells at 36 h posttransfection were lysed, and GST-LKB1 was affinity purified and assayed for the ability to activate the heterotrimeric AMPK complex expressed in E. coli , as described in Materials and Methods . Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the SD for a single triplicate experiment). Affinity-purified GST-LKB1 preparation (upper panel), as well as cell extracts (lower panel), was immunoblotted with the indicated antibodies.

Techniques Used: Activity Assay, Binding Assay, Fluorescence, Mutagenesis, Staining, SDS Page, Affinity Purification, Purification, Transfection, Construct

71) Product Images from "Molecular determinants that mediate selective activation of p38 MAP kinase isoforms"

Article Title: Molecular determinants that mediate selective activation of p38 MAP kinase isoforms

Journal: The EMBO Journal

doi: 10.1093/emboj/19.6.1301

Fig. 2. Binding of p38α and p38β2 to MAP kinase kinases. ( A ) Primary sequences of the N–terminal domain of MKK3, MKK3b, MKK6 and deletion mutants (Δ) are aligned. Residues that are identical to MKK3b are indicated with a dot (.). The residues of MKK3b (LRI) and MKK6 (LKI) deleted in MKK3bΔ and MKK6Δ are indicated in bold. The deleted residues are indicated with a dash (–). Basic residues are indicated by asterisks. ( B ) Activated GST-tagged MKK3, K6(1–18)–K3 or K6(1–82)–K3 were co-transfected with an empty vector (Control), Flag-tagged p38α or Flag-tagged p38β2 in COS7 cells. The activated MKKs were constructed by replacing the two sites of activating phosphorylation with glutamic acid residues. Protein expression was monitored by immunoblot analysis of cell extracts. The GST–MKK fusion proteins were isolated from the cell extracts by incubation with glutathione–Sepharose. The co-precipitation of p38α and p38β2 with the MKK was examined by immunoblot analysis with an antibody to the Flag epitope. ( C and D ) The interaction of Flag-tagged p38α and p38β2 with GST-tagged activated MKK3, MKK3b and MKK3bΔ (C) or MKK6 and MKK6Δ (D) co-expressed in COS7 cells was examined using the methods described in (B). The activated MKKs were constructed by replacing the two sites of activating phosphorylation with glutamic acid residues.
Figure Legend Snippet: Fig. 2. Binding of p38α and p38β2 to MAP kinase kinases. ( A ) Primary sequences of the N–terminal domain of MKK3, MKK3b, MKK6 and deletion mutants (Δ) are aligned. Residues that are identical to MKK3b are indicated with a dot (.). The residues of MKK3b (LRI) and MKK6 (LKI) deleted in MKK3bΔ and MKK6Δ are indicated in bold. The deleted residues are indicated with a dash (–). Basic residues are indicated by asterisks. ( B ) Activated GST-tagged MKK3, K6(1–18)–K3 or K6(1–82)–K3 were co-transfected with an empty vector (Control), Flag-tagged p38α or Flag-tagged p38β2 in COS7 cells. The activated MKKs were constructed by replacing the two sites of activating phosphorylation with glutamic acid residues. Protein expression was monitored by immunoblot analysis of cell extracts. The GST–MKK fusion proteins were isolated from the cell extracts by incubation with glutathione–Sepharose. The co-precipitation of p38α and p38β2 with the MKK was examined by immunoblot analysis with an antibody to the Flag epitope. ( C and D ) The interaction of Flag-tagged p38α and p38β2 with GST-tagged activated MKK3, MKK3b and MKK3bΔ (C) or MKK6 and MKK6Δ (D) co-expressed in COS7 cells was examined using the methods described in (B). The activated MKKs were constructed by replacing the two sites of activating phosphorylation with glutamic acid residues.

Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Construct, Expressing, Isolation, Incubation, FLAG-tag

Fig. 5. Inhibition of p38β2 activity by peptide competition. ( A ) The primary sequence of synthetic peptides corresponding to the native (wt-pep) or mutated (gly-pep) N–terminal region of MKK3b is shown. The mutated peptide was prepared by replacing the residues of MKK3b (LRI), indicated in bold, with glycine. ( B ) Purified bacterially expressed GST–p38α was bound to glutathione–Sepharose and incubated with 100 μM wild-type or mutated MKK3b peptide. Purified activated MKK3, MKK3b or MKK6 were incubated with the immobilized GST–p38α in kinase buffer with ATP for 20 min. The GST–p38α was washed with kinase buffer and the p38α activity was measured using ATF2 and [γ- 32 P]ATP as the substrates. The phosphorylated ATF2 was detected after SDS–PAGE by auto- radiography and was quantitated by PhosphorImager analysis. The p38α activity is presented as relative protein kinase activity. ( C – F ) Purified bacterially expressed GST–p38β2 was bound to glutathione–Sepharose and incubated with increasing concentrations of the wild-type (C and E) or mutated (D and F) MKK3b peptide. Purified activated MKK3b (C and D) or MKK6 (E and F) were incubated with the immobilized GST–p38β2 in kinase buffer with ATP for 20 min. The GST–p38β2 activity was measured as described in (B).
Figure Legend Snippet: Fig. 5. Inhibition of p38β2 activity by peptide competition. ( A ) The primary sequence of synthetic peptides corresponding to the native (wt-pep) or mutated (gly-pep) N–terminal region of MKK3b is shown. The mutated peptide was prepared by replacing the residues of MKK3b (LRI), indicated in bold, with glycine. ( B ) Purified bacterially expressed GST–p38α was bound to glutathione–Sepharose and incubated with 100 μM wild-type or mutated MKK3b peptide. Purified activated MKK3, MKK3b or MKK6 were incubated with the immobilized GST–p38α in kinase buffer with ATP for 20 min. The GST–p38α was washed with kinase buffer and the p38α activity was measured using ATF2 and [γ- 32 P]ATP as the substrates. The phosphorylated ATF2 was detected after SDS–PAGE by auto- radiography and was quantitated by PhosphorImager analysis. The p38α activity is presented as relative protein kinase activity. ( C – F ) Purified bacterially expressed GST–p38β2 was bound to glutathione–Sepharose and incubated with increasing concentrations of the wild-type (C and E) or mutated (D and F) MKK3b peptide. Purified activated MKK3b (C and D) or MKK6 (E and F) were incubated with the immobilized GST–p38β2 in kinase buffer with ATP for 20 min. The GST–p38β2 activity was measured as described in (B).

Techniques Used: Inhibition, Activity Assay, Sequencing, Purification, Incubation, SDS Page

72) Product Images from "ATP and MO25? Regulate the Conformational State of the STRAD? Pseudokinase and Activation of the LKB1 Tumour Suppressor"

Article Title: ATP and MO25? Regulate the Conformational State of the STRAD? Pseudokinase and Activation of the LKB1 Tumour Suppressor

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1000126

Mutation of MO25α concave surface residues abolishes STRADα and LKB1 binding. (A and B) The indicated constructs of GST-STRADα and Myc-MO25α were expressed in 293 cells. Cells 36 h post-transfection were lysed, and GST-STRADα was purified with glutathione-Sepharose. The purified GST-STRADα preparation (upper panels), as well as the cell extracts (lower panels), was immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments. Dotted line indicates the junction of two gels. AL, activation loop. (C and D) Two hundred ninety-three cells were cotransfected with the indicated constructs of GST-LKB1, Flag-STRADα, and Myc-MO25α. Cells 36 h post-transfection were lysed, and GST-LKB1 was purified and assayed for its ability to phosphorylate the LKBtide peptide. Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the standard deviation for one experiment). Affinity-purified GST-LKB1 preparation (upper panel), as well as cell extracts (lower panel), was immunoblotted with the indicated antibodies.
Figure Legend Snippet: Mutation of MO25α concave surface residues abolishes STRADα and LKB1 binding. (A and B) The indicated constructs of GST-STRADα and Myc-MO25α were expressed in 293 cells. Cells 36 h post-transfection were lysed, and GST-STRADα was purified with glutathione-Sepharose. The purified GST-STRADα preparation (upper panels), as well as the cell extracts (lower panels), was immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments. Dotted line indicates the junction of two gels. AL, activation loop. (C and D) Two hundred ninety-three cells were cotransfected with the indicated constructs of GST-LKB1, Flag-STRADα, and Myc-MO25α. Cells 36 h post-transfection were lysed, and GST-LKB1 was purified and assayed for its ability to phosphorylate the LKBtide peptide. Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the standard deviation for one experiment). Affinity-purified GST-LKB1 preparation (upper panel), as well as cell extracts (lower panel), was immunoblotted with the indicated antibodies.

Techniques Used: Mutagenesis, Binding Assay, Construct, Transfection, Purification, Activation Assay, Standard Deviation, Affinity Purification

Interaction of ATP and MO25α with STRADα controls LKB1 activity. (A) The structure of the ATP binding site of STRADα in which the key interacting residues are emphasized. (B) Fluorescence emission spectra (excitation 410 nm) of TNP-ATP (5 µM) bound to wild-type and mutant forms of STRADα (2 µM) and/or wild-type MO25α (2 µM). A reference cuvette containing only TNP-ATP (5 µM) was subtracted as background. A Coomassie Blue-stained SDS-PAGE gel of each form of STRADα analysed is shown (GGK = G76D+G78D+K197E, GGR = G76D+G78D+R215E, and GGKR = G76D+G78D+K197E+R215E). (C) Wild-type GST-LKB1 and indicated forms of Flag-STRADα were expressed in 293 cells in the absence of MO25α. Cells at 36 h posttransfection were lysed and GST-LKB1 affinity purified on glutathione-Sepharose. The purified GST-LKB1 preparation (upper panel), as well as the cell extracts (lower panel), was immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments. Dotted line indicates where the gel was cut. (D and E) 293 cells were co-transfected with the indicated constructs of GST-LKB1, Flag-STRADα, and Myc-MO25α. Cells at 36 h posttransfection were lysed, and GST-LKB1 was affinity purified and assayed for the ability to activate the heterotrimeric AMPK complex expressed in E. coli , as described in Materials and Methods . Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the SD for a single triplicate experiment). Affinity-purified GST-LKB1 preparation (upper panel), as well as cell extracts (lower panel), was immunoblotted with the indicated antibodies.
Figure Legend Snippet: Interaction of ATP and MO25α with STRADα controls LKB1 activity. (A) The structure of the ATP binding site of STRADα in which the key interacting residues are emphasized. (B) Fluorescence emission spectra (excitation 410 nm) of TNP-ATP (5 µM) bound to wild-type and mutant forms of STRADα (2 µM) and/or wild-type MO25α (2 µM). A reference cuvette containing only TNP-ATP (5 µM) was subtracted as background. A Coomassie Blue-stained SDS-PAGE gel of each form of STRADα analysed is shown (GGK = G76D+G78D+K197E, GGR = G76D+G78D+R215E, and GGKR = G76D+G78D+K197E+R215E). (C) Wild-type GST-LKB1 and indicated forms of Flag-STRADα were expressed in 293 cells in the absence of MO25α. Cells at 36 h posttransfection were lysed and GST-LKB1 affinity purified on glutathione-Sepharose. The purified GST-LKB1 preparation (upper panel), as well as the cell extracts (lower panel), was immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments. Dotted line indicates where the gel was cut. (D and E) 293 cells were co-transfected with the indicated constructs of GST-LKB1, Flag-STRADα, and Myc-MO25α. Cells at 36 h posttransfection were lysed, and GST-LKB1 was affinity purified and assayed for the ability to activate the heterotrimeric AMPK complex expressed in E. coli , as described in Materials and Methods . Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the SD for a single triplicate experiment). Affinity-purified GST-LKB1 preparation (upper panel), as well as cell extracts (lower panel), was immunoblotted with the indicated antibodies.

Techniques Used: Activity Assay, Binding Assay, Fluorescence, Mutagenesis, Staining, SDS Page, Affinity Purification, Purification, Transfection, Construct

73) Product Images from "The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways"

Article Title: The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200705021

Identification of motifs required for direct EspF and SNX9 interactions. (A) Glutathione-Sepharose pull-down with 10 μg of GST-EspF (residues 48–206) mixed with the [ 35 S]-methionine proteins indicated (left diagram). Autoradiograph of GST pulldown (left) and of 1/20th input of 35 S-labeled NCK1, NCK2, SNX9 (residues 1–111), and GRB2 is shown (right). (B) HEK293A cells were cotransfected with EGFP-EspF and V5-tagged proteins indicated (left diagram). Anti-GFP immunoprecipitations (IP) were probed by V5 immunoblot (IB) (left). Cell lysates were probed by V5 or GFP immunoblot to show input levels. (C) Logos plot of the SNX9 binding consensus sequence derived by phage display experiments (left). Alignment of 13 unique SNX9 binding sequences used to derive the consensus is shown. The invariant arginine (blue) and highly conserved residues (gray) are highlighted. (D) Peptide array analysis of the SNX9-binding sites on EspF. Top: diagram of EspF residues 1–166 used for the peptide scanning experiments. Middle: ultraviolet (UV) illumination shows the qualitative amount of each peptide synthesized (top). Bottom: solid-phase binding of 35 S-SNX9 to 15-mer EspF peptides was assessed by autoradiography. An alignment of EspF-binding peptides from two SNX9 binding series is shown. (E) Saturation binding curves were generated with increasing concentrations of GST-SNX9-SH3 (left) to a fixed concentration of EspF peptide by fluorescence polarization (see Materials and methods). (F) HEK293A cells were cotransfected with EGFP-EspF or triple mutant EGFP-EspF-D3 (top diagram) and V5-tagged SNX9. Anti-GFP immunoprecipitations (IP) were probed by V5 immunoblot (IB) (top panel). Cell lysates were probed by V5 or GFP immunoblot to show input levels (bottom two panels).
Figure Legend Snippet: Identification of motifs required for direct EspF and SNX9 interactions. (A) Glutathione-Sepharose pull-down with 10 μg of GST-EspF (residues 48–206) mixed with the [ 35 S]-methionine proteins indicated (left diagram). Autoradiograph of GST pulldown (left) and of 1/20th input of 35 S-labeled NCK1, NCK2, SNX9 (residues 1–111), and GRB2 is shown (right). (B) HEK293A cells were cotransfected with EGFP-EspF and V5-tagged proteins indicated (left diagram). Anti-GFP immunoprecipitations (IP) were probed by V5 immunoblot (IB) (left). Cell lysates were probed by V5 or GFP immunoblot to show input levels. (C) Logos plot of the SNX9 binding consensus sequence derived by phage display experiments (left). Alignment of 13 unique SNX9 binding sequences used to derive the consensus is shown. The invariant arginine (blue) and highly conserved residues (gray) are highlighted. (D) Peptide array analysis of the SNX9-binding sites on EspF. Top: diagram of EspF residues 1–166 used for the peptide scanning experiments. Middle: ultraviolet (UV) illumination shows the qualitative amount of each peptide synthesized (top). Bottom: solid-phase binding of 35 S-SNX9 to 15-mer EspF peptides was assessed by autoradiography. An alignment of EspF-binding peptides from two SNX9 binding series is shown. (E) Saturation binding curves were generated with increasing concentrations of GST-SNX9-SH3 (left) to a fixed concentration of EspF peptide by fluorescence polarization (see Materials and methods). (F) HEK293A cells were cotransfected with EGFP-EspF or triple mutant EGFP-EspF-D3 (top diagram) and V5-tagged SNX9. Anti-GFP immunoprecipitations (IP) were probed by V5 immunoblot (IB) (top panel). Cell lysates were probed by V5 or GFP immunoblot to show input levels (bottom two panels).

Techniques Used: Autoradiography, Labeling, Binding Assay, Sequencing, Derivative Assay, Peptide Microarray, Synthesized, Generated, Concentration Assay, Fluorescence, Mutagenesis

SNX9 is the major binding partner for EspF in polarized epithelial cells. (A) TER measurements of polarized T84 colonic epithelial cells infected with EPEC and the indicated EspF mutants. The average change in TER in three independent experiments is shown. (B and C) Fluorescence microscopy of MDCK cells uninfected (B) or infected with EPEC for 4 h (C). Tight junction morphology was detected by anti-occludin immunocytochemistry. (D) Schematic of TAP tagged EspF is shown. Western blot of cellular lysates collected from stable MDCK cells expressing TAP-flag-EspF or mutant TAP-flag-EspF-D3. Anti-flag was used to probe EspF expression (top blot) and actin was used as a protein loading control (bottom blot). (E and F) Fluorescence microscopy of MDCK cells expressing TAP-EspF and TAP-EspF-D3. Tight junction morphology was detected by anti-occludin immunocytochemistry. (G) MDCK parental or Tap-EspF cellular lysates were incubated with anti-flag agarose and the resulting immuno-complexes were subjected to SDS-PAGE and stained with Coomassie. Proteins identified by mass spectrometry are indicated. “NS” designates nonspecific interacting proteins and “IgG LC” is the immunoglobulin light chain. (H) Anti-SNX9 immunofluorescence microscopy of CaCo 2 cells infected with the indicated EPEC strains for 3 h. Boxed area is a 4× magnification of the area indicated. Cellular borders are outlined. Bar = 15 μm. (I) Immunoblot of EspF from wild-type EPEC (Wt) or EPEC espF − strains carrying plasmids encoding wild-type EspF ( pespf ) or mutant EspF-D3 ( pespf-D3 ) tagged with the myc epitope. Type III secreted EspF harvested from media supernatants (top) and from whole bacterial lysates (bottom) are shown. Concentrations of IPTG are indicated below.
Figure Legend Snippet: SNX9 is the major binding partner for EspF in polarized epithelial cells. (A) TER measurements of polarized T84 colonic epithelial cells infected with EPEC and the indicated EspF mutants. The average change in TER in three independent experiments is shown. (B and C) Fluorescence microscopy of MDCK cells uninfected (B) or infected with EPEC for 4 h (C). Tight junction morphology was detected by anti-occludin immunocytochemistry. (D) Schematic of TAP tagged EspF is shown. Western blot of cellular lysates collected from stable MDCK cells expressing TAP-flag-EspF or mutant TAP-flag-EspF-D3. Anti-flag was used to probe EspF expression (top blot) and actin was used as a protein loading control (bottom blot). (E and F) Fluorescence microscopy of MDCK cells expressing TAP-EspF and TAP-EspF-D3. Tight junction morphology was detected by anti-occludin immunocytochemistry. (G) MDCK parental or Tap-EspF cellular lysates were incubated with anti-flag agarose and the resulting immuno-complexes were subjected to SDS-PAGE and stained with Coomassie. Proteins identified by mass spectrometry are indicated. “NS” designates nonspecific interacting proteins and “IgG LC” is the immunoglobulin light chain. (H) Anti-SNX9 immunofluorescence microscopy of CaCo 2 cells infected with the indicated EPEC strains for 3 h. Boxed area is a 4× magnification of the area indicated. Cellular borders are outlined. Bar = 15 μm. (I) Immunoblot of EspF from wild-type EPEC (Wt) or EPEC espF − strains carrying plasmids encoding wild-type EspF ( pespf ) or mutant EspF-D3 ( pespf-D3 ) tagged with the myc epitope. Type III secreted EspF harvested from media supernatants (top) and from whole bacterial lysates (bottom) are shown. Concentrations of IPTG are indicated below.

Techniques Used: Binding Assay, Infection, Fluorescence, Microscopy, Immunocytochemistry, Western Blot, Expressing, Mutagenesis, Incubation, SDS Page, Staining, Mass Spectrometry, Immunofluorescence

74) Product Images from "Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro"

Article Title: Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004074

Syntaxin 4 disrupts the interaction of Munc18c with VAMP2. (A) 5 µg of M18c was immobilised on Ni-NTA agarose and incubated overnight with an excess (10 µg) of Sx4 cytosolic domain (residues 1–273) (final concentration 0.7 µM Munc18c, 3.3 µM Sx4 in the initial incubation). After extensive washing, the pre-formed complex was subsequently incubated with increasing concentrations of VAMP2 cytosolic domain (residues 1–92) as indicated. Upper panel shows a Coomassie stained gel of the input proteins; lower panel shows an immunoblot of the beads probed with anti-Munc18c, or stained with Coomassie to show levels of Sx4. (B) 5 µg of M18c was immobilised on Ni-NTA agarose and incubated overnight with an excess (10 µg) of VAMP2 cytosolic domain (final concentration 0.7 µM Munc18c, 7 µM VAMP2 in the initial incubation). The upper panel (input) shows an immunoblot analysis of these beads after extensive washing confirming the presence of both Munc18c and VAMP2. This preformed complex was then incubated alone or with 250 µg of Sx4 cytosolic domain or (as a control) 250 µg Sx16 cytosolic domain (residues 1–269) (both t-SNAREs at a final concentration 16.7 µM), as indicated. Immunoblot analysis was again used to determine the amount of Munc18c and VAMP2 that remained bound following this challenge (lower panel). Data shown are representative of three experiments of this type.
Figure Legend Snippet: Syntaxin 4 disrupts the interaction of Munc18c with VAMP2. (A) 5 µg of M18c was immobilised on Ni-NTA agarose and incubated overnight with an excess (10 µg) of Sx4 cytosolic domain (residues 1–273) (final concentration 0.7 µM Munc18c, 3.3 µM Sx4 in the initial incubation). After extensive washing, the pre-formed complex was subsequently incubated with increasing concentrations of VAMP2 cytosolic domain (residues 1–92) as indicated. Upper panel shows a Coomassie stained gel of the input proteins; lower panel shows an immunoblot of the beads probed with anti-Munc18c, or stained with Coomassie to show levels of Sx4. (B) 5 µg of M18c was immobilised on Ni-NTA agarose and incubated overnight with an excess (10 µg) of VAMP2 cytosolic domain (final concentration 0.7 µM Munc18c, 7 µM VAMP2 in the initial incubation). The upper panel (input) shows an immunoblot analysis of these beads after extensive washing confirming the presence of both Munc18c and VAMP2. This preformed complex was then incubated alone or with 250 µg of Sx4 cytosolic domain or (as a control) 250 µg Sx16 cytosolic domain (residues 1–269) (both t-SNAREs at a final concentration 16.7 µM), as indicated. Immunoblot analysis was again used to determine the amount of Munc18c and VAMP2 that remained bound following this challenge (lower panel). Data shown are representative of three experiments of this type.

Techniques Used: Incubation, Concentration Assay, Staining

Munc18c binds directly to the cytosolic domains of Syntaxin 4 and VAMP2. (A) 5 µg of either GST, or GST fused to the cytosolic domain of Sx4, VAMP2, VAMP 4 or VAMP 8, immobilised on glutathione Sepharose were incubated overnight at 4 C with 5 µg His-tagged Munc18c in a volume of 500 µl. After extensive washing in binding buffer, SDS-PAGE and immunoblot analysis was used to determine which of the GST proteins Munc18c had bound to. Upper panel represents a Coomassie stained gel of input proteins; lower panel shows an immunoblot for bound Munc18c (B) The ability of Munc18c to bind to a version of GST-VAMP2 harbouring only the SNARE motif of the v-SNARE was assessed as in (A). Data are representative of four experiments of this type.
Figure Legend Snippet: Munc18c binds directly to the cytosolic domains of Syntaxin 4 and VAMP2. (A) 5 µg of either GST, or GST fused to the cytosolic domain of Sx4, VAMP2, VAMP 4 or VAMP 8, immobilised on glutathione Sepharose were incubated overnight at 4 C with 5 µg His-tagged Munc18c in a volume of 500 µl. After extensive washing in binding buffer, SDS-PAGE and immunoblot analysis was used to determine which of the GST proteins Munc18c had bound to. Upper panel represents a Coomassie stained gel of input proteins; lower panel shows an immunoblot for bound Munc18c (B) The ability of Munc18c to bind to a version of GST-VAMP2 harbouring only the SNARE motif of the v-SNARE was assessed as in (A). Data are representative of four experiments of this type.

Techniques Used: Incubation, Binding Assay, SDS Page, Staining

75) Product Images from "O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release"

Article Title: O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release

Journal: The EMBO Journal

doi: 10.1038/emboj.2012.8

TAB1 is O -GlcNAcylated on Ser395. ( A ) Recombinant TAB1 was O -GlcNAcylated in vitro with recombinant hOGT. The samples were denatured in LDS (lithium dodecyl sulphate), subjected to SDS–PAGE and immunoblotted with a generic O -GlcNAc antibody CTD110.6. Total protein was detected with a total TAB1 antibody (TAB1) as a loading control. ( B ) In vitro O -GlcNAcylated TAB1 was subjected to enzymatic labelling using galactosyltransferase (mGalT1) labelling and UDP-GalNAz, before reacting with biotin alkyne for detection with HRP. The samples were denatured in LDS, subjected to SDS–PAGE and probed with horseradish peroxidase-conjugated streptavidin (Extravidin-HRP). ( C ) In vivo O -GlcNAcylation of TAB1 was detected by immunoprecipitating the endogenous TAB1 from IL-1R cells (treated with or without GlcNAcstatin—1 μM) using an antibody directed against TAB1. A non-specific IgG was included as a control. Immunoprecipitates were denatured in LDS, subjected to SDS–PAGE and immunoblotted with generic O -GlcNAc antibody CTD110.6 and then with the TAB1 antibody for loading controls. Treating the cells with the potent and selective OGA inhibitor GlcNAcstatin further increased TAB1 O -GlcNAcylation at a concentration of 1 μM. ( D ) TAB1 was immunoprecipitated, from cells treated with or without GlcNAcstatin (1 μM), using the TAB1 antibody. One fraction of the reaction was treated with Cp OGA for 30 min at room temperature, denatured in LDS, followed by immunoblotting with the generic O -GlcNAc antibody CTD110.6. A parallel set of immunoprecipitated samples were denatured in LDS and subjected to western blotting with antibody pre-incubated with N -acetylglucosamine, which blocks the O -GlcNAc signal on TAB1. Lower panel shows the corresponding Ponceau-stained membrane before western blotting. ( E ) WT TAB1, the S391A/S395A/S396A TAB1 triple mutant and the S391A, S395A and S396A TAB1 single mutants were transfected into IL-1R cells. After 24 h, the cells were treated with 1 μM of GlcNAcstatin for 16 h and the GSt–TAB1 was pulled out using glutathione-sepharose beads. The samples were denatured in LDS, subjected to SDS–PAGE and immunoblotted with the generic O -GlcNAc antibody CTD110.6 and then with a total TAB1 antibody (TAB1) as a loading control. ( F , G ) LC-MS/MS CID ( F ) and ETD ( G ) site mapping of the TAB1 O -GlcNAc modification site. In vitro O-GlcNAcylated TAB1 was digested with trypsin and subjected to LC–MS. The tryptic peptide VYPVSVPYSSAQSTSK (Mw calc =1901.9 Da) containing a HexNAc (+203.1 Da) was detected after 25.5 min as [M+HexNAc+2H] 2+ m / z 951.904. The observed fragment ions are indicated, in case of the ETD experiment allowing definition of S395 as the site of O -GlcNAc modification. Figure source data can be found in Supplementary data .
Figure Legend Snippet: TAB1 is O -GlcNAcylated on Ser395. ( A ) Recombinant TAB1 was O -GlcNAcylated in vitro with recombinant hOGT. The samples were denatured in LDS (lithium dodecyl sulphate), subjected to SDS–PAGE and immunoblotted with a generic O -GlcNAc antibody CTD110.6. Total protein was detected with a total TAB1 antibody (TAB1) as a loading control. ( B ) In vitro O -GlcNAcylated TAB1 was subjected to enzymatic labelling using galactosyltransferase (mGalT1) labelling and UDP-GalNAz, before reacting with biotin alkyne for detection with HRP. The samples were denatured in LDS, subjected to SDS–PAGE and probed with horseradish peroxidase-conjugated streptavidin (Extravidin-HRP). ( C ) In vivo O -GlcNAcylation of TAB1 was detected by immunoprecipitating the endogenous TAB1 from IL-1R cells (treated with or without GlcNAcstatin—1 μM) using an antibody directed against TAB1. A non-specific IgG was included as a control. Immunoprecipitates were denatured in LDS, subjected to SDS–PAGE and immunoblotted with generic O -GlcNAc antibody CTD110.6 and then with the TAB1 antibody for loading controls. Treating the cells with the potent and selective OGA inhibitor GlcNAcstatin further increased TAB1 O -GlcNAcylation at a concentration of 1 μM. ( D ) TAB1 was immunoprecipitated, from cells treated with or without GlcNAcstatin (1 μM), using the TAB1 antibody. One fraction of the reaction was treated with Cp OGA for 30 min at room temperature, denatured in LDS, followed by immunoblotting with the generic O -GlcNAc antibody CTD110.6. A parallel set of immunoprecipitated samples were denatured in LDS and subjected to western blotting with antibody pre-incubated with N -acetylglucosamine, which blocks the O -GlcNAc signal on TAB1. Lower panel shows the corresponding Ponceau-stained membrane before western blotting. ( E ) WT TAB1, the S391A/S395A/S396A TAB1 triple mutant and the S391A, S395A and S396A TAB1 single mutants were transfected into IL-1R cells. After 24 h, the cells were treated with 1 μM of GlcNAcstatin for 16 h and the GSt–TAB1 was pulled out using glutathione-sepharose beads. The samples were denatured in LDS, subjected to SDS–PAGE and immunoblotted with the generic O -GlcNAc antibody CTD110.6 and then with a total TAB1 antibody (TAB1) as a loading control. ( F , G ) LC-MS/MS CID ( F ) and ETD ( G ) site mapping of the TAB1 O -GlcNAc modification site. In vitro O-GlcNAcylated TAB1 was digested with trypsin and subjected to LC–MS. The tryptic peptide VYPVSVPYSSAQSTSK (Mw calc =1901.9 Da) containing a HexNAc (+203.1 Da) was detected after 25.5 min as [M+HexNAc+2H] 2+ m / z 951.904. The observed fragment ions are indicated, in case of the ETD experiment allowing definition of S395 as the site of O -GlcNAc modification. Figure source data can be found in Supplementary data .

Techniques Used: Recombinant, In Vitro, SDS Page, In Vivo, Concentration Assay, Immunoprecipitation, Western Blot, Incubation, Staining, Mutagenesis, Transfection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification

O -GlcNAcylation of TAB1 affects activation of TAK1 and phosphorylation of its downstream targets IκBα and JNK1/2. ( A ) IL-1α or NaCl-induced activation of TAK1 in WT and S395A TAB1 transfected Tab1 −/− MEFs. At 36 h post-transfection, MEFs were serum starved for 6 h, and then stimulated for 5 and 15 min with 10 ng/ml IL-1α or 0.5 M NaCl. The TAK1 complexes were pulled down from the cell extracts (1 mg of protein extract) using glutathione-sepharose beads, and TAK1 activity assays were performed (as described in the Materials and methods section) in addition to immunoblotting as described below in ( B ). The data are expressed as the relative increase in TAK1 activity of the IL-1/NaCl-stimulated samples compared with the basal activity of the unstimulated control samples. Error bars denote standard deviation, determined from three independent experiments. ( B ) In parallel to the experiments in ( A ), TAK1 complexes were denatured in LDS, subjected to SDS–PAGE and immunoblotted with a phospho-specific antibody that recognizes TAK1 autophosphorylation at Thr187 (pT187) and with a further antibody that recognizes all forms of TAK1. O -GlcNAcylation of TAB1 was detected with the site-specific O -GlcNAc antibody (gS395) versus a total TAB1 antibody control (TAB1). ( C ) In all, 30 μg of the cell lysates from the samples obtained as in ( A ) was immunoblotted for phosphorylated Iκβα p-Iκβα and total Iκβα. ( D ) Densitometry for IκBα phosphorylation after normalization against total TAB1 levels. The data shown are the average of three independent experiments with error bars denoting standard deviation. ( E ) WT and S395A TAB1 were transfected in Tab1 −/− MEFs. At 36 h post-transfection, MEFs were serum starved for 6 h, and then stimulated for 5 or 15 min with 0.5 M NaCl. The TAK1 complexes were pulled down from the cell extracts (1 mg of protein extract) using glutathione-sepharose beads and taken for kinase assays ( A ) in addition to immunoblotting. For immunoblotting, the samples were denatured in LDS, subjected to SDS–PAGE and immunoblotted with a phospho-specific antibody that recognizes TAK1 autophosphorylation at Thr187 (pT187) and with a further antibody that recognizes all forms of TAK1. O -GlcNAcylation of TAB1 was detected with the site-specific O -GlcNAc antibody (gS395) versus a total TAB1 antibody control (TAB1). ( F ) In all, 30 μg of the cell lysates from the samples obtained as in ( E ) was immunoblotted for phosphorylated JNK1/2 (p-JNK1/2) and total JNK1/2. ( G ) Densitometry for JNK1/2 phosphorylation after normalization for total JNK1/2. The data shown are the average of minimum of three independent experiments with error bars denoting standard deviation. Figure source data can be found in Supplementary data .
Figure Legend Snippet: O -GlcNAcylation of TAB1 affects activation of TAK1 and phosphorylation of its downstream targets IκBα and JNK1/2. ( A ) IL-1α or NaCl-induced activation of TAK1 in WT and S395A TAB1 transfected Tab1 −/− MEFs. At 36 h post-transfection, MEFs were serum starved for 6 h, and then stimulated for 5 and 15 min with 10 ng/ml IL-1α or 0.5 M NaCl. The TAK1 complexes were pulled down from the cell extracts (1 mg of protein extract) using glutathione-sepharose beads, and TAK1 activity assays were performed (as described in the Materials and methods section) in addition to immunoblotting as described below in ( B ). The data are expressed as the relative increase in TAK1 activity of the IL-1/NaCl-stimulated samples compared with the basal activity of the unstimulated control samples. Error bars denote standard deviation, determined from three independent experiments. ( B ) In parallel to the experiments in ( A ), TAK1 complexes were denatured in LDS, subjected to SDS–PAGE and immunoblotted with a phospho-specific antibody that recognizes TAK1 autophosphorylation at Thr187 (pT187) and with a further antibody that recognizes all forms of TAK1. O -GlcNAcylation of TAB1 was detected with the site-specific O -GlcNAc antibody (gS395) versus a total TAB1 antibody control (TAB1). ( C ) In all, 30 μg of the cell lysates from the samples obtained as in ( A ) was immunoblotted for phosphorylated Iκβα p-Iκβα and total Iκβα. ( D ) Densitometry for IκBα phosphorylation after normalization against total TAB1 levels. The data shown are the average of three independent experiments with error bars denoting standard deviation. ( E ) WT and S395A TAB1 were transfected in Tab1 −/− MEFs. At 36 h post-transfection, MEFs were serum starved for 6 h, and then stimulated for 5 or 15 min with 0.5 M NaCl. The TAK1 complexes were pulled down from the cell extracts (1 mg of protein extract) using glutathione-sepharose beads and taken for kinase assays ( A ) in addition to immunoblotting. For immunoblotting, the samples were denatured in LDS, subjected to SDS–PAGE and immunoblotted with a phospho-specific antibody that recognizes TAK1 autophosphorylation at Thr187 (pT187) and with a further antibody that recognizes all forms of TAK1. O -GlcNAcylation of TAB1 was detected with the site-specific O -GlcNAc antibody (gS395) versus a total TAB1 antibody control (TAB1). ( F ) In all, 30 μg of the cell lysates from the samples obtained as in ( E ) was immunoblotted for phosphorylated JNK1/2 (p-JNK1/2) and total JNK1/2. ( G ) Densitometry for JNK1/2 phosphorylation after normalization for total JNK1/2. The data shown are the average of minimum of three independent experiments with error bars denoting standard deviation. Figure source data can be found in Supplementary data .

Techniques Used: Activation Assay, Transfection, Activity Assay, Standard Deviation, SDS Page

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Article Snippet: His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE Healthcare), GST-fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2 , 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE Healthcare). .. Recombinant GST-Tsr2 was immobilized in PBSKMT (high—500 mM NaCl addition and low—no NaCl addition salt conditions) on Glutathione Sepharose (GE Healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , and eS26ΔESS1ΔESS2-FLAG for 1 h at 4 °C.

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE healthcare), GST fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2, 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE healthcare). .. Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: .. For glutathione S -transferase (GST) pulldown assays, GST-Xic1, GST-hp21, and GST-hp27 fusion proteins (5 μg) were bound to glutathione-Sepharose 4B (GE Healthcare) and incubated with [35 S]methionine-labeled XCdt2 (4 μl) for 1.5 h at 23°C. .. For GST-XCdt21-400 , GST-XCdt2401-710 , and GST-XPCNA, beads were incubated with purified MBP-Xic1 or XPCNA (5, 15, 25, or 50 μg).

Mass Spectrometry:

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿
Article Snippet: The lysate was cleared by centrifugation for 1 h at 20,000 rpm in an SS 34 rotor and incubated for 2 h at 4°C with glutathione-Sepharose (Amersham Biosciences) preequilibrated in PBS-KMT. .. Integrity of the protein N termini was confirmed by protein digestion with trypsin (Promega), peptide separation by high-pressure liquid chromatography (UltiMate 3000 high-pressure liquid chromatography system; Dionex), and detection of the N-terminal peptides via mass spectrometry analysis (4000 Q TRAP mass spectrometer; Applied Biosystems/MDS Sciex).

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: Paragraph title: Analysis of proteins by mass spectrometry. ... The GST and GST-Xic1 proteins (5 μg) were coupled to glutathione-Sepharose 4B (GE Healthcare) and incubated in 250 μl of LSS for 1 h at 4°C.

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: For glutathione S -transferase (GST) pulldown assays, GST-Xic1, GST-hp21, and GST-hp27 fusion proteins (5 μg) were bound to glutathione-Sepharose 4B (GE Healthcare) and incubated with [35 S]methionine-labeled XCdt2 (4 μl) for 1.5 h at 23°C. .. Protein bands from Coomassie blue staining were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Voyager-DE Pro; Applied Biosystems) as described previously ( ).

Acrylamide Gel Assay:

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: The GST and GST-Xic1 proteins (5 μg) were coupled to glutathione-Sepharose 4B (GE Healthcare) and incubated in 250 μl of LSS for 1 h at 4°C. .. The GST or GST-Xic1 binding proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the corresponding acrylamide gel lanes were sliced into 8 pieces and digested with 10 ng/μl trypsin (Promega) at 37°C for 18 h. The peptides were extracted in 5% formic acid and 50% acetonitrile and dried in a SpeedVac (Savant).

Western Blot:

Article Title: Inhibition of PACT-Mediated Activation of PKR by the Herpes Simplex Virus Type 1 Us11 Protein
Article Snippet: Proteins to be tested for interaction with Us11C were independently mixed with 1 μg of GST or GST-Us11C in binding buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1% Triton X-100, 20% glycerol, 100 U of aprotinin/ml, 0.2 mM PMSF) or high-salt buffer containing 25 μl of glutathione-Sepharose 4B (Amersham Pharmacia) and placed on a rotating wheel for 2 h at 4o C. After binding, the beads were washed six times with 500 μl of fresh buffer. .. The proteins interacting with the GST-containing protein were analyzed by Western blotting for FLAG, histidine, or PACT domain 3.

Transformation Assay:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: Expression and purification of recombinant proteins The E. coli strain BL21(DE3)LysS was transformed with plasmids encoding full-length or truncated GST-Rrp6p polypeptides and grown up at 30°C in LB medium containing ampicillin and chloramphenicol to an OD600 of 0.5. .. Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione.

High Performance Liquid Chromatography:

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿
Article Snippet: The lysate was cleared by centrifugation for 1 h at 20,000 rpm in an SS 34 rotor and incubated for 2 h at 4°C with glutathione-Sepharose (Amersham Biosciences) preequilibrated in PBS-KMT. .. Integrity of the protein N termini was confirmed by protein digestion with trypsin (Promega), peptide separation by high-pressure liquid chromatography (UltiMate 3000 high-pressure liquid chromatography system; Dionex), and detection of the N-terminal peptides via mass spectrometry analysis (4000 Q TRAP mass spectrometer; Applied Biosystems/MDS Sciex).

Protease Inhibitor:

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿
Article Snippet: After induction with 1 mM IPTG (isopropyl-β- d -thiogalactopyranoside) for 3 h at 15°C, cells were lysed by sonication in phosphate-buffered saline (PBS)-KMT (PBS supplemented with 1 mM MgCl2 , 3 mM KCl and 0.1% Tween 20) containing the Complete protease inhibitor mixture (added according to the manufacturer's instructions) (Roche). .. The lysate was cleared by centrifugation for 1 h at 20,000 rpm in an SS 34 rotor and incubated for 2 h at 4°C with glutathione-Sepharose (Amersham Biosciences) preequilibrated in PBS-KMT.

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: .. For each reaction, 20 μl (bed volume) glutathione-Sepharose 4B (GE Healthcare) loaded with GST or GST fusion proteins was washed twice in 700 μl binding buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM DTT, 10% glycerol, 0.5% Triton X-100, EDTA-free protease inhibitor cocktail set III). .. Acid-extracted histones from one-half of a 15-cm dish of H1299 cells in 300 μl binding buffer were subjected to one 10-min and two 5-min centrifugations (20,000 × g ) to remove insoluble debris.

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: The affinity matrix was prepared by washing glutathione-Sepharose 4B (GE Healthcare) consecutively in 10 bed volumes of equilibration buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM MgCl2 ), 10 bed volumes of blocking buffer (equilibration buffer with 2% bovine serum albumin [BSA]) (1 h under rotation), and another 10 volumes of equilibration buffer. .. After that, 1.25 ml equilibrated matrix per 1,000-ml culture volume and the supernatant from the bacterial lysate were combined and rotated for 2 h. The sample was then applied to a 10-ml Pierce centrifuge column (Thermo Scientific), and the matrix was washed consecutively with 50 bed volumes of low-salt wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM DTT, 1% Triton X-100, 1 mM EDTA, cOmplete EDTA-free protease inhibitor cocktail), 50 bed volumes of high-salt wash buffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 1 mM DTT, 1% Triton X-100, 1 mM EDTA, cOmplete EDTA-free protease inhibitor cocktail), and another 50 bed volumes of low-salt wash buffer.

SDS Page:

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: The GST and GST-Xic1 proteins (5 μg) were coupled to glutathione-Sepharose 4B (GE Healthcare) and incubated in 250 μl of LSS for 1 h at 4°C. .. The GST or GST-Xic1 binding proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the corresponding acrylamide gel lanes were sliced into 8 pieces and digested with 10 ng/μl trypsin (Promega) at 37°C for 18 h. The peptides were extracted in 5% formic acid and 50% acetonitrile and dried in a SpeedVac (Savant).

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: For glutathione S -transferase (GST) pulldown assays, GST-Xic1, GST-hp21, and GST-hp27 fusion proteins (5 μg) were bound to glutathione-Sepharose 4B (GE Healthcare) and incubated with [35 S]methionine-labeled XCdt2 (4 μl) for 1.5 h at 23°C. .. The beads were washed with NETN buffer (50 mM Tris, 250 mM NaCl, 5 mM EDTA at pH 7.5, and 0.1% NP-40) and subjected to SDS-PAGE and phosphorimager analysis or Coomassie blue staining.

Sonication:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: Cell lysates were prepared by sonication in 20 mM HEPES pH 7.6, 300 mM NaCl, 10 mM imidazole pH 7.6 and clarified by centrifugation at 15 000g for 30 min. .. Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione.

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿
Article Snippet: After induction with 1 mM IPTG (isopropyl-β- d -thiogalactopyranoside) for 3 h at 15°C, cells were lysed by sonication in phosphate-buffered saline (PBS)-KMT (PBS supplemented with 1 mM MgCl2 , 3 mM KCl and 0.1% Tween 20) containing the Complete protease inhibitor mixture (added according to the manufacturer's instructions) (Roche). .. The lysate was cleared by centrifugation for 1 h at 20,000 rpm in an SS 34 rotor and incubated for 2 h at 4°C with glutathione-Sepharose (Amersham Biosciences) preequilibrated in PBS-KMT.

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: Following addition of lysozyme (150 μg/ml) and benzonase (25 U/ml), the suspension was sonicated five times for 1 min using a Branson model 450 sonifier (duty cycle, 80%; output control, 2) to facilitate cell lysis. .. The affinity matrix was prepared by washing glutathione-Sepharose 4B (GE Healthcare) consecutively in 10 bed volumes of equilibration buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM MgCl2 ), 10 bed volumes of blocking buffer (equilibration buffer with 2% bovine serum albumin [BSA]) (1 h under rotation), and another 10 volumes of equilibration buffer.

Affinity Purification:

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2
Article Snippet: His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE Healthcare), GST-fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2 , 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE Healthcare). .. Recombinant GST-Tsr2 was immobilized in PBSKMT (high—500 mM NaCl addition and low—no NaCl addition salt conditions) on Glutathione Sepharose (GE Healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , and eS26ΔESS1ΔESS2-FLAG for 1 h at 4 °C.

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE healthcare), GST fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2, 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE healthcare). .. Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2
Article Snippet: .. His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE Healthcare), GST-fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2 , 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE Healthcare). .. Recombinant GST-Tsr2 was immobilized in PBSKMT (high—500 mM NaCl addition and low—no NaCl addition salt conditions) on Glutathione Sepharose (GE Healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , and eS26ΔESS1ΔESS2-FLAG for 1 h at 4 °C.

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: .. His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE healthcare), GST fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2, 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE healthcare). .. GST-tagged importins, His6 -taggged importins and RanGTP (His6 -Gsp1Q71L-GTP) were expressed and purified as previously described ( ; ; ).

Recombinant:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: Paragraph title: Expression and purification of recombinant proteins ... Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione.

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿
Article Snippet: Paragraph title: Recombinant protein expression and purification. ... The lysate was cleared by centrifugation for 1 h at 20,000 rpm in an SS 34 rotor and incubated for 2 h at 4°C with glutathione-Sepharose (Amersham Biosciences) preequilibrated in PBS-KMT.

Article Title: Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13
Article Snippet: .. Recombinant proteins were purified on glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, N.J.) or Ni-nitrilotriacetic acid agarose (Qiagen). .. Kap13 and pQE30-Ran were expressed in the strain M15[pREP4] (Qiagen).

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2
Article Snippet: .. Recombinant GST-Tsr2 was immobilized in PBSKMT (high—500 mM NaCl addition and low—no NaCl addition salt conditions) on Glutathione Sepharose (GE Healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , and eS26ΔESS1ΔESS2-FLAG for 1 h at 4 °C. .. After incubation, the immobilized GST-proteins were washed three times with 1 ml of PBSKMT 4 °C.

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: .. Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C. .. After incubation, the immobilized GST-proteins were washed three times with PBSKMT 4°C.

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: .. Protein and RNA analyses Recombinant protein-binding assays were performed by mixing lysates from cells expressing one protein with pull-downs of the partner protein on glutathione-sepharose or Ni-NTA superflow beads. .. After extensively washing the beads in lysis buffer, retained proteins were eluted, resolved by SDS–PAGE and visualized by staining with Coomassie blue G250 or transferred to nylon membrane and decorated with penta-His monoclonal antibodies (Qiagen) or anti-GST antiserum (Sigma).

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2
Article Snippet: Paragraph title: Recombinant protein expression and binding assays ... His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE Healthcare), GST-fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2 , 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE Healthcare).

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: Paragraph title: Recombinant protein expression and binding assays ... His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE healthcare), GST fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2, 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE healthcare).

GST Pulldown Assay:

Article Title: Inhibition of PACT-Mediated Activation of PKR by the Herpes Simplex Virus Type 1 Us11 Protein
Article Snippet: Paragraph title: GST pulldown assay. ... Proteins to be tested for interaction with Us11C were independently mixed with 1 μg of GST or GST-Us11C in binding buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1% Triton X-100, 20% glycerol, 100 U of aprotinin/ml, 0.2 mM PMSF) or high-salt buffer containing 25 μl of glutathione-Sepharose 4B (Amersham Pharmacia) and placed on a rotating wheel for 2 h at 4o C. After binding, the beads were washed six times with 500 μl of fresh buffer.

Nucleic Acid Electrophoresis:

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: The GST and GST-Xic1 proteins (5 μg) were coupled to glutathione-Sepharose 4B (GE Healthcare) and incubated in 250 μl of LSS for 1 h at 4°C. .. The GST or GST-Xic1 binding proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the corresponding acrylamide gel lanes were sliced into 8 pieces and digested with 10 ng/μl trypsin (Promega) at 37°C for 18 h. The peptides were extracted in 5% formic acid and 50% acetonitrile and dried in a SpeedVac (Savant).

Ion Exchange Chromatography:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione. .. His( )-Rrp47p was further purified by ion exchange chromatography and gel filtration.

Purification:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: Paragraph title: Expression and purification of recombinant proteins ... Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione.

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿
Article Snippet: Paragraph title: Recombinant protein expression and purification. ... The lysate was cleared by centrifugation for 1 h at 20,000 rpm in an SS 34 rotor and incubated for 2 h at 4°C with glutathione-Sepharose (Amersham Biosciences) preequilibrated in PBS-KMT.

Article Title: Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13
Article Snippet: .. Recombinant proteins were purified on glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, N.J.) or Ni-nitrilotriacetic acid agarose (Qiagen). .. Kap13 and pQE30-Ran were expressed in the strain M15[pREP4] (Qiagen).

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2
Article Snippet: His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE Healthcare), GST-fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2 , 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE Healthcare). .. Recombinant GST-Tsr2 was immobilized in PBSKMT (high—500 mM NaCl addition and low—no NaCl addition salt conditions) on Glutathione Sepharose (GE Healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , and eS26ΔESS1ΔESS2-FLAG for 1 h at 4 °C.

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: GST-tagged importins, His6 -taggged importins and RanGTP (His6 -Gsp1Q71L-GTP) were expressed and purified as previously described ( ; ; ). .. Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2
Article Snippet: .. His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE Healthcare), GST-fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2 , 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE Healthcare). .. Recombinant GST-Tsr2 was immobilized in PBSKMT (high—500 mM NaCl addition and low—no NaCl addition salt conditions) on Glutathione Sepharose (GE Healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , and eS26ΔESS1ΔESS2-FLAG for 1 h at 4 °C.

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: .. His6 -tagged proteins were affinity purified in 50 mM Hepes pH 7.5, 50 mM NaCl, 10% glycerol using Ni-NTA Agarose (GE healthcare), GST fusion proteins were purified in PBSKMT (150 mM NaCl, 25 mM sodium phosphate, 3 mM KCl, 1 mM MgCl2, 0.1% Tween, pH 7.3) using Glutathione Sepharose (GE healthcare). .. GST-tagged importins, His6 -taggged importins and RanGTP (His6 -Gsp1Q71L-GTP) were expressed and purified as previously described ( ; ; ).

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: For glutathione S -transferase (GST) pulldown assays, GST-Xic1, GST-hp21, and GST-hp27 fusion proteins (5 μg) were bound to glutathione-Sepharose 4B (GE Healthcare) and incubated with [35 S]methionine-labeled XCdt2 (4 μl) for 1.5 h at 23°C. .. For GST-XCdt21-400 , GST-XCdt2401-710 , and GST-XPCNA, beads were incubated with purified MBP-Xic1 or XPCNA (5, 15, 25, or 50 μg).

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: Paragraph title: Protein production, purification, and analysis. ... The affinity matrix was prepared by washing glutathione-Sepharose 4B (GE Healthcare) consecutively in 10 bed volumes of equilibration buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM MgCl2 ), 10 bed volumes of blocking buffer (equilibration buffer with 2% bovine serum albumin [BSA]) (1 h under rotation), and another 10 volumes of equilibration buffer.

Affinity Chromatography:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione. .. The eluate from the Ni-NTA affinity chromatography was diluted 10-fold with 20 mM HEPES pH 7.6 300 mM NaCl to reduce the imidazole concentration and then mixed with SP-sepharose resin.

Blocking Assay:

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: .. The affinity matrix was prepared by washing glutathione-Sepharose 4B (GE Healthcare) consecutively in 10 bed volumes of equilibration buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM MgCl2 ), 10 bed volumes of blocking buffer (equilibration buffer with 2% bovine serum albumin [BSA]) (1 h under rotation), and another 10 volumes of equilibration buffer. .. After that, 1.25 ml equilibrated matrix per 1,000-ml culture volume and the supernatant from the bacterial lysate were combined and rotated for 2 h. The sample was then applied to a 10-ml Pierce centrifuge column (Thermo Scientific), and the matrix was washed consecutively with 50 bed volumes of low-salt wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM DTT, 1% Triton X-100, 1 mM EDTA, cOmplete EDTA-free protease inhibitor cocktail), 50 bed volumes of high-salt wash buffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 1 mM DTT, 1% Triton X-100, 1 mM EDTA, cOmplete EDTA-free protease inhibitor cocktail), and another 50 bed volumes of low-salt wash buffer.

Lysis:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: .. Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione. .. His( )-Rrp47p was further purified by ion exchange chromatography and gel filtration.

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: After that, 1 ml IP lysis buffer was added, and the matrix was washed five times in the same buffer. .. For each reaction, 20 μl (bed volume) glutathione-Sepharose 4B (GE Healthcare) loaded with GST or GST fusion proteins was washed twice in 700 μl binding buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM DTT, 10% glycerol, 0.5% Triton X-100, EDTA-free protease inhibitor cocktail set III).

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: Following addition of lysozyme (150 μg/ml) and benzonase (25 U/ml), the suspension was sonicated five times for 1 min using a Branson model 450 sonifier (duty cycle, 80%; output control, 2) to facilitate cell lysis. .. The affinity matrix was prepared by washing glutathione-Sepharose 4B (GE Healthcare) consecutively in 10 bed volumes of equilibration buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM MgCl2 ), 10 bed volumes of blocking buffer (equilibration buffer with 2% bovine serum albumin [BSA]) (1 h under rotation), and another 10 volumes of equilibration buffer.

Liquid Chromatography:

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: The GST and GST-Xic1 proteins (5 μg) were coupled to glutathione-Sepharose 4B (GE Healthcare) and incubated in 250 μl of LSS for 1 h at 4°C. .. The tryptic peptides were separated by UltiMate Nano liquid chromatography (LC) systems (LC Packings) and sequenced using a QStar mass spectrometer (Applied Biosystems) as described previously ( ).

Binding Assay:

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2
Article Snippet: Paragraph title: Recombinant protein expression and binding assays ... Recombinant GST-Tsr2 was immobilized in PBSKMT (high—500 mM NaCl addition and low—no NaCl addition salt conditions) on Glutathione Sepharose (GE Healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , and eS26ΔESS1ΔESS2-FLAG for 1 h at 4 °C.

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: Paragraph title: Recombinant protein expression and binding assays ... Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

Article Title: Nuclear Localization Signal and Protein Context both Mediate Importin ? Specificity of Nuclear Import Substrates ▿
Article Snippet: Paragraph title: Importin α binding assay. ... GST fusion proteins (approximately 100 pmol) were immobilized on glutathione-Sepharose (Amersham Biosciences) preequilibrated in import buffer (specified above) for 1 h at 4°C.

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: .. For each reaction, 20 μl (bed volume) glutathione-Sepharose 4B (GE Healthcare) loaded with GST or GST fusion proteins was washed twice in 700 μl binding buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM DTT, 10% glycerol, 0.5% Triton X-100, EDTA-free protease inhibitor cocktail set III). .. Acid-extracted histones from one-half of a 15-cm dish of H1299 cells in 300 μl binding buffer were subjected to one 10-min and two 5-min centrifugations (20,000 × g ) to remove insoluble debris.

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: The GST and GST-Xic1 proteins (5 μg) were coupled to glutathione-Sepharose 4B (GE Healthcare) and incubated in 250 μl of LSS for 1 h at 4°C. .. The GST or GST-Xic1 binding proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the corresponding acrylamide gel lanes were sliced into 8 pieces and digested with 10 ng/μl trypsin (Promega) at 37°C for 18 h. The peptides were extracted in 5% formic acid and 50% acetonitrile and dried in a SpeedVac (Savant).

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: .. To examine the binding between XPCNA and Xic1, GST-PCNA fusion proteins (5 μg) were bound to glutathione-Sepharose 4B and incubated with [35 S]methionine-labeled Xic1-NPIP mutants (4 μl) for 1.5 h at 23°C. .. The beads were washed with NETN buffer and subjected to SDS-PAGE and PhosphorImager analysis.

Article Title: Inhibition of PACT-Mediated Activation of PKR by the Herpes Simplex Virus Type 1 Us11 Protein
Article Snippet: .. Proteins to be tested for interaction with Us11C were independently mixed with 1 μg of GST or GST-Us11C in binding buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1% Triton X-100, 20% glycerol, 100 U of aprotinin/ml, 0.2 mM PMSF) or high-salt buffer containing 25 μl of glutathione-Sepharose 4B (Amersham Pharmacia) and placed on a rotating wheel for 2 h at 4o C. After binding, the beads were washed six times with 500 μl of fresh buffer. .. The proteins interacting with the GST-containing protein were analyzed by Western blotting for FLAG, histidine, or PACT domain 3.

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: Paragraph title: In vitro binding assays. ... For glutathione S -transferase (GST) pulldown assays, GST-Xic1, GST-hp21, and GST-hp27 fusion proteins (5 μg) were bound to glutathione-Sepharose 4B (GE Healthcare) and incubated with [35 S]methionine-labeled XCdt2 (4 μl) for 1.5 h at 23°C.

In Vitro:

Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2
Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT (high—500 mM NaCl addition and low—no NaCl addition salt conditions) on Glutathione Sepharose (GE Healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , and eS26ΔESS1ΔESS2-FLAG for 1 h at 4 °C. .. The in vitro binding studies between recombinant eS26FLAG , eS26ΔESS1-FLAG , eS26ΔESS2-FLAG , eS26ΔESS1ΔESS2-FLAG , Tsr2, Tsr2:eS26 complex, and yeast importins as GST-fusion proteins were performed as previously described .

Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly
Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C. .. The in vitro binding studies between recombinant eS26FLAG , eS26D33NFLAG, eS26C77WFLAG, Tsr2, Tsr2:eS26 complex and yeast importins as GST-fusion proteins were performed as previously described ( ).

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: Paragraph title: In vitro binding assays. ... For glutathione S -transferase (GST) pulldown assays, GST-Xic1, GST-hp21, and GST-hp27 fusion proteins (5 μg) were bound to glutathione-Sepharose 4B (GE Healthcare) and incubated with [35 S]methionine-labeled XCdt2 (4 μl) for 1.5 h at 23°C.

Protein Binding:

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: Paragraph title: Protein binding and competition analysis. ... For each reaction, 20 μl (bed volume) glutathione-Sepharose 4B (GE Healthcare) loaded with GST or GST fusion proteins was washed twice in 700 μl binding buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM DTT, 10% glycerol, 0.5% Triton X-100, EDTA-free protease inhibitor cocktail set III).

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: .. Protein and RNA analyses Recombinant protein-binding assays were performed by mixing lysates from cells expressing one protein with pull-downs of the partner protein on glutathione-sepharose or Ni-NTA superflow beads. .. After extensively washing the beads in lysis buffer, retained proteins were eluted, resolved by SDS–PAGE and visualized by staining with Coomassie blue G250 or transferred to nylon membrane and decorated with penta-His monoclonal antibodies (Qiagen) or anti-GST antiserum (Sigma).

Concentration Assay:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione. .. The eluate from the Ni-NTA affinity chromatography was diluted 10-fold with 20 mM HEPES pH 7.6 300 mM NaCl to reduce the imidazole concentration and then mixed with SP-sepharose resin.

Staining:

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: For glutathione S -transferase (GST) pulldown assays, GST-Xic1, GST-hp21, and GST-hp27 fusion proteins (5 μg) were bound to glutathione-Sepharose 4B (GE Healthcare) and incubated with [35 S]methionine-labeled XCdt2 (4 μl) for 1.5 h at 23°C. .. The beads were washed with NETN buffer (50 mM Tris, 250 mM NaCl, 5 mM EDTA at pH 7.5, and 0.1% NP-40) and subjected to SDS-PAGE and phosphorimager analysis or Coomassie blue staining.

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    GE Healthcare glutathione sepharose
    The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione <t>Sepharose</t> and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014
    Glutathione Sepharose, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare glutathione sepharose beads
    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with <t>glutathione-sepharose</t> beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.
    Glutathione Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Mutagenesis, Binding Assay, Transformation Assay, Western Blot, Fluorescence In Situ Hybridization, Labeling, Staining, Recombinant, Incubation, SDS Page, Expressing, Fluorescence, Microscopy

    Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Sequencing, Incubation, SDS Page, Staining, Western Blot, Stable Transfection, In Vitro

    RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Incubation, SDS Page, Staining, Western Blot

    eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Mutagenesis, Western Blot, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Staining, Sedimentation, Centrifugation, Agarose Gel Electrophoresis, Northern Blot

    Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Purification, Isolation, Agarose Gel Electrophoresis, Northern Blot, Silver Staining, Western Blot

    GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Sedimentation, Western Blot, Recombinant, Incubation, SDS Page, Staining, Transformation Assay, Binding Assay, Expressing, Fluorescence, Microscopy, Mutagenesis

    eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Recombinant, Incubation, Purification, SDS Page, Staining, Western Blot

    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Recombinant, Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Protein Concentration, Silver Staining, Western Blot, Incubation, Imaging

    GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Blocking Assay, Silver Staining, Quantitation Assay, Western Blot, Imaging, Incubation

    GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Mass Spectrometry, Isolation, Liquid Chromatography with Mass Spectroscopy

    GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Mass Spectrometry, Cell Culture

    GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Expressing, Lysis, Incubation, SDS Page, Staining, Silver Staining, Mutagenesis, Infection, Activity Assay, Reporter Assay

    GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Staining, Binding Assay, Transfection, Expressing, Construct, Positive Control, SDS Page, Silver Staining, Infection, Activity Assay, Reporter Assay, Western Blot, Imaging