Structured Review

GE Healthcare glutathione sepharose 4b
α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on <t>glutathione-sepharose</t> beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
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1) Product Images from "Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation"

Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.02.011

α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
Figure Legend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

Techniques Used: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

2) Product Images from "Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes"

Article Title: Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks243

In vitro interaction of CryB and AppA. Western blots of 12% SDS–PAGE from [glutathione S transferase (GST)- and MBP-] pull-down assays using a CryB-specific antibody (A–D) or a LOV-specific antibody (E). ( A ) AppA-MBP protein bound to amylose–agarose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( B ) Incubation of cell lysate from R.s. Δ cryB (pRK pufcryB ) with amylose–agarose. ( C ) GST-AppAΔN bound to glutathione-sepharose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( D ) GST-SCHIC bound to glutathione–sepharose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( E ) AppA-MBP protein bound to amylose–agarose and incubated with cell lysate from R.s. 2.4.1(pRK puflov ). F, cell lysate flow through; W, washing fractions (same volume as F); E, elution fractions (same volume as F).
Figure Legend Snippet: In vitro interaction of CryB and AppA. Western blots of 12% SDS–PAGE from [glutathione S transferase (GST)- and MBP-] pull-down assays using a CryB-specific antibody (A–D) or a LOV-specific antibody (E). ( A ) AppA-MBP protein bound to amylose–agarose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( B ) Incubation of cell lysate from R.s. Δ cryB (pRK pufcryB ) with amylose–agarose. ( C ) GST-AppAΔN bound to glutathione-sepharose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( D ) GST-SCHIC bound to glutathione–sepharose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( E ) AppA-MBP protein bound to amylose–agarose and incubated with cell lysate from R.s. 2.4.1(pRK puflov ). F, cell lysate flow through; W, washing fractions (same volume as F); E, elution fractions (same volume as F).

Techniques Used: In Vitro, Western Blot, SDS Page, Incubation, Flow Cytometry

3) Product Images from "Cupidin, an Isoform of Homer/Vesl, Interacts with the Actin Cytoskeleton and Activated Rho Family Small GTPases and Is Expressed in Developing Mouse Cerebellar Granule Cells"

Article Title: Cupidin, an Isoform of Homer/Vesl, Interacts with the Actin Cytoskeleton and Activated Rho Family Small GTPases and Is Expressed in Developing Mouse Cerebellar Granule Cells

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.19-19-08389.1999

Cupidin interacts with F-actin and mGluR1α. A , F-actin binding to Cupidin in a cosedimentation assay. GST, GST-CPD/N, and GST-CPD/C were incubated with (+) or without (−) F-actin prepared from chicken skeletal muscles and then centrifuged. Equivalent protein amounts of the supernatant ( S ) and pellet ( P ) fractions were separated by SDS-PAGE and stained with Coomassie Brilliant Blue ( CBB ). A representative result of five independent experiments is shown here. The arrows indicate the position of actin, and the arrowheads represent each GST-fusion protein. B , Binding of Cupidin to mGluR1α in a pull down assay. The S1 fraction prepared from P7 mouse cerebellum or cerebrum was incubated with GST-CPD/N protein and then immobilized onto glutathione-Sepharose beads. After extensive washing, GST-CPD/N-bound proteins were extracted with SDS-PAGE sample buffer and were analyzed by Western blotting using an anti-mGluR1α polyclonal antibody. Lane 1 , Input (the same amounts of lysates used for the assay were loaded); lane 2 , eluate from a GST-bound column; lane 3 , eluate from a GST-CPD/N bound column. C , Coimmunoprecipitation of both mGluR1 and actin from P7 mouse cerebellar lysates using the anti-CPD antibody. The immunoprecipitates obtained with either the preimmune serum ( lane 2 ) or the affinity-purified anti-CPD polyclonal antibody were examined by Western blotting with the indicated antibodies (anti-CPD, anti-actin, and anti-mGluR1α). Lane 1 is the detergent extract of P7 mouse cerebellum. The arrow indicates Cupidin signal, and the asterisk indicates the heavy chain of IgG. D , Primary-cultured mouse cerebellar granule neurons at 7 DIV were triple-stained with an anti-CPD polyclonal antibody (FITC; a ), Texas Red-phalloidin ( b ), and an anti-synaptophysin monoclonal antibody (Cy5; c ) and observed by confocal microscopy. d is a superimposed composite images of a–c using three pseudocolors ( green for CPD, red for phalloidin, and blue for synaptophysin). Arrows indicate representative positions at which the three pseudocolors overlapped. Scale bar, 10 μm.
Figure Legend Snippet: Cupidin interacts with F-actin and mGluR1α. A , F-actin binding to Cupidin in a cosedimentation assay. GST, GST-CPD/N, and GST-CPD/C were incubated with (+) or without (−) F-actin prepared from chicken skeletal muscles and then centrifuged. Equivalent protein amounts of the supernatant ( S ) and pellet ( P ) fractions were separated by SDS-PAGE and stained with Coomassie Brilliant Blue ( CBB ). A representative result of five independent experiments is shown here. The arrows indicate the position of actin, and the arrowheads represent each GST-fusion protein. B , Binding of Cupidin to mGluR1α in a pull down assay. The S1 fraction prepared from P7 mouse cerebellum or cerebrum was incubated with GST-CPD/N protein and then immobilized onto glutathione-Sepharose beads. After extensive washing, GST-CPD/N-bound proteins were extracted with SDS-PAGE sample buffer and were analyzed by Western blotting using an anti-mGluR1α polyclonal antibody. Lane 1 , Input (the same amounts of lysates used for the assay were loaded); lane 2 , eluate from a GST-bound column; lane 3 , eluate from a GST-CPD/N bound column. C , Coimmunoprecipitation of both mGluR1 and actin from P7 mouse cerebellar lysates using the anti-CPD antibody. The immunoprecipitates obtained with either the preimmune serum ( lane 2 ) or the affinity-purified anti-CPD polyclonal antibody were examined by Western blotting with the indicated antibodies (anti-CPD, anti-actin, and anti-mGluR1α). Lane 1 is the detergent extract of P7 mouse cerebellum. The arrow indicates Cupidin signal, and the asterisk indicates the heavy chain of IgG. D , Primary-cultured mouse cerebellar granule neurons at 7 DIV were triple-stained with an anti-CPD polyclonal antibody (FITC; a ), Texas Red-phalloidin ( b ), and an anti-synaptophysin monoclonal antibody (Cy5; c ) and observed by confocal microscopy. d is a superimposed composite images of a–c using three pseudocolors ( green for CPD, red for phalloidin, and blue for synaptophysin). Arrows indicate representative positions at which the three pseudocolors overlapped. Scale bar, 10 μm.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining, Pull Down Assay, Western Blot, Affinity Purification, Cell Culture, Confocal Microscopy

4) Product Images from "The Leber Congenital Amaurosis Protein AIPL1 Functions as Part of a Chaperone Heterocomplex"

Article Title: The Leber Congenital Amaurosis Protein AIPL1 Functions as Part of a Chaperone Heterocomplex

Journal: Investigative ophthalmology & visual science

doi: 10.1167/iovs.07-1576

Characterization of AIPL1-chaperone interactions using in vitro protein-binding assays. ( A ) Affinity pull-down of endogenous Hsp90 and Hsp70 with GST-AIPL1. GST and GST-AIPL1 were affinity purified on glutathione Sepharose 4B ( lower ; Coomassie stain).
Figure Legend Snippet: Characterization of AIPL1-chaperone interactions using in vitro protein-binding assays. ( A ) Affinity pull-down of endogenous Hsp90 and Hsp70 with GST-AIPL1. GST and GST-AIPL1 were affinity purified on glutathione Sepharose 4B ( lower ; Coomassie stain).

Techniques Used: In Vitro, Protein Binding, Affinity Purification, Staining

5) Product Images from "TMEM74 promotes tumor cell survival by inducing autophagy via interactions with ATG16L1 and ATG9A"

Article Title: TMEM74 promotes tumor cell survival by inducing autophagy via interactions with ATG16L1 and ATG9A

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.370

TMEM74 associates with ATG16L1 via its C-terminal and influences the interaction between ATG5 and ATG16L1. ( a ) Schematic representations of WT ATG16L1 and its mutants: ATG16L1(1–320), and ATG16L1 △(1–320) . ( b ) HeLa cells were co-transfected with GFP-ATG16L1 and mCherry-TMEM74 for 24 h, Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( c ) HeLa cells were co-transfected with GFP-TMEM74 and mCherry-ATG16L1 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, ATG16L1 was detected in the washed beads using an anti-ATG16L1 IgG by western blotting. ( d ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG16L1, GFP-ATG16L1 was detected in the washed beads by western blotting. ( e , f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG16L1(1–320), or GFP-ATG16L1 △(1–320) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 were detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG16L1 for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, ATG5-ATG12 complex pulled down was detected in the immunoprecipitates using anti-ATG5 by western blotting. Quantification of ATG5-ATG12 pulled down relative to GFP-ATG16L1 was shown as column. Data are means±S.D. of three experiments. * P
Figure Legend Snippet: TMEM74 associates with ATG16L1 via its C-terminal and influences the interaction between ATG5 and ATG16L1. ( a ) Schematic representations of WT ATG16L1 and its mutants: ATG16L1(1–320), and ATG16L1 △(1–320) . ( b ) HeLa cells were co-transfected with GFP-ATG16L1 and mCherry-TMEM74 for 24 h, Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( c ) HeLa cells were co-transfected with GFP-TMEM74 and mCherry-ATG16L1 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, ATG16L1 was detected in the washed beads using an anti-ATG16L1 IgG by western blotting. ( d ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG16L1, GFP-ATG16L1 was detected in the washed beads by western blotting. ( e , f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG16L1(1–320), or GFP-ATG16L1 △(1–320) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 were detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG16L1 for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, ATG5-ATG12 complex pulled down was detected in the immunoprecipitates using anti-ATG5 by western blotting. Quantification of ATG5-ATG12 pulled down relative to GFP-ATG16L1 was shown as column. Data are means±S.D. of three experiments. * P

Techniques Used: Transfection, Western Blot, Incubation

TMEM74 associates with ATG9A via its N-terminal and influences the interaction between ATG9 and WIPI1. ( a ) HeLa cells were co-transfected with GFP-ATG9A and mCherry-TMEM74 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( b ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG9A, GFP-ATG9A was detected in the washed beads using an anti-GFP IgG by western blotting. ( c ) Schematic representations of WT-ATG9A and its mutants: ATG9A(1–495), and ATG9A △(1–495) , and ATG9A △(153–289) . ( d – f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG9A(1–495), GFP-ATG9A △(1–495) , or GFP-ATG9A △(153–289) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 was detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG9A for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, WIPI1 pulled down was detected in the immunoprecipitates using the anti-WIPI1 antibody by western blotting. Quantification of WIPI1 pulled down relative to GFP-ATG9A was shown as column. Data are means±S.D. of three experiments. * P
Figure Legend Snippet: TMEM74 associates with ATG9A via its N-terminal and influences the interaction between ATG9 and WIPI1. ( a ) HeLa cells were co-transfected with GFP-ATG9A and mCherry-TMEM74 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( b ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG9A, GFP-ATG9A was detected in the washed beads using an anti-GFP IgG by western blotting. ( c ) Schematic representations of WT-ATG9A and its mutants: ATG9A(1–495), and ATG9A △(1–495) , and ATG9A △(153–289) . ( d – f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG9A(1–495), GFP-ATG9A △(1–495) , or GFP-ATG9A △(153–289) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 was detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG9A for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, WIPI1 pulled down was detected in the immunoprecipitates using the anti-WIPI1 antibody by western blotting. Quantification of WIPI1 pulled down relative to GFP-ATG9A was shown as column. Data are means±S.D. of three experiments. * P

Techniques Used: Transfection, Western Blot, Incubation

6) Product Images from "Rad51 protects nascent DNA from Mre11 dependent degradation and promotes continuous DNA synthesis"

Article Title: Rad51 protects nascent DNA from Mre11 dependent degradation and promotes continuous DNA synthesis

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.1927

Rad51 and PCNA modifications in DNA replication and ssDNA gap accumulation. (A) Rad51 and PCNA requirement for replication of untreated and MMS-treated DNA. Sperm nuclei were incubated in 10 μl egg extract with α 32 P-dATP for the indicated times in the presence or absence of 0.7 mg ml −1 GST or GST-BRC4 and MMS (− or +), and 0.2 mg ml −1 of recombinant wild type PCNA (WT) or mutated PCNA (K164R). Replication products were resolved on 1% (w/v) alkaline agarose gel and subjected to autoradiography. (B) The signal intensities obtained in (A) were quantified and reported on the graph. The experiments shown represent a typical result. (C) Gap labelling procedure using T4 DNA polymerase. Replicating genomic DNA was isolated and used as a template for gap-filling assay using T4 DNA polymerase. The labelled nascent molecules extended by T4 were then resolved on alkaline agarose gel. (D) Untreated (−MMS) and MMS treated (+MMS) sperm nuclei were incubated in 10 μl of egg extract in the presence of GST or GST-BRC4 for 60 min (1–4). Untreated sperm nuclei were incubated for 40, 60 or 80 min in the presence of PCNA-WT or PCNA-K164R (5–10). Genomic DNA was isolated and subjected to the gap labelling reaction followed by autoradiography. Exposure times are equivalent for the 2 gels although kinetic profile starts at 40 minutes in 5–10. The graph shows the relative fold increase in optical density measured for each lane taking as reference untreated chromatin recovered at 60 minutes. The experiment shows a typical result.
Figure Legend Snippet: Rad51 and PCNA modifications in DNA replication and ssDNA gap accumulation. (A) Rad51 and PCNA requirement for replication of untreated and MMS-treated DNA. Sperm nuclei were incubated in 10 μl egg extract with α 32 P-dATP for the indicated times in the presence or absence of 0.7 mg ml −1 GST or GST-BRC4 and MMS (− or +), and 0.2 mg ml −1 of recombinant wild type PCNA (WT) or mutated PCNA (K164R). Replication products were resolved on 1% (w/v) alkaline agarose gel and subjected to autoradiography. (B) The signal intensities obtained in (A) were quantified and reported on the graph. The experiments shown represent a typical result. (C) Gap labelling procedure using T4 DNA polymerase. Replicating genomic DNA was isolated and used as a template for gap-filling assay using T4 DNA polymerase. The labelled nascent molecules extended by T4 were then resolved on alkaline agarose gel. (D) Untreated (−MMS) and MMS treated (+MMS) sperm nuclei were incubated in 10 μl of egg extract in the presence of GST or GST-BRC4 for 60 min (1–4). Untreated sperm nuclei were incubated for 40, 60 or 80 min in the presence of PCNA-WT or PCNA-K164R (5–10). Genomic DNA was isolated and subjected to the gap labelling reaction followed by autoradiography. Exposure times are equivalent for the 2 gels although kinetic profile starts at 40 minutes in 5–10. The graph shows the relative fold increase in optical density measured for each lane taking as reference untreated chromatin recovered at 60 minutes. The experiment shows a typical result.

Techniques Used: Incubation, Recombinant, Agarose Gel Electrophoresis, Autoradiography, Isolation

7) Product Images from "Subnuclear domain proteins in cancer cells support the functions of RUNX2 in the DNA damage response"

Article Title: Subnuclear domain proteins in cancer cells support the functions of RUNX2 in the DNA damage response

Journal: Journal of Cell Science

doi: 10.1242/jcs.160051

Interaction of RUNX2 with RUVBL2, INTS3 and BAZ1B. (A) The functional domains of RUNX2, RUVBL2, INTS3 and BAZ1B, and the location of peptide sequences identified by mass spectrometry. The peptide fragment identified by mass spectrometry is indicated as a closed bar. Functional domains in each peptide are indicated. RHD, Runt homolog domain; NMTS, nuclear matrix targeting sequences; AAA, ATPase associated with a variety of cellular activities; KD, kinase domain; DDT, DNA binding homeobox and different transcription factors; PHD, plant homeodomain; BRD, bromodomain. (B) Co-immunoprecipitation of RUNX2 with interacting proteins was analyzed by western blotting. To detect RUNX2–RUVBL2, RUNX2–INTS3 or RUNX2–BAZ1B endogenous interactions, 5 mg of whole-cell lysates from Saos2 or U2OS cells were immunoprecipitated (IP) with 5 µg of anti-RUNX2 antibodies or 5 µg of normal rabbit IgG as a negative control. Immunoprecipitation products were then analyzed by western blotting, using anti-RUVBL2, anti-INTS3 or anti-BAZ1B antibodies. Note that no clear immunoprecipitation products were seen using anti-INTS3 antibodies and the results are not shown. (C) Co-immunoprecipitation of FLAG–RUVBL2 protein with full-length RUNX2 [wildtype (WT), amino acids 1–528] or C-terminally deleted mutant (ΔC, amino acids 1–376). U2OS cells were transiently co-transfected with a FLAG–RUVBL2 expression construct and either full-length or C-terminally deleted RUNX2 construct. Whole-cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma). Washed beads were subjected to SDS-PAGE and analyzed by western blotting (WB) using specific antibodies against the indicated proteins. Asterisks (*) mark bands caused by nonspecific interactions. (D) Bacterially expressed GST (‘G’), GST fused to the Runt homolog domain of RUNX2 (amino acids 107–241; GST-R) or GST fused to the C-terminus of RUNX2 (amino acids 240–528; GST-C) proteins were immobilized on glutathione beads and incubated with whole-cell lysates from Saos2 cells. After extensive washing, proteins bound to the beads were eluted in protein sample buffer and analyzed by western blotting with antibodies against the indicated proteins.
Figure Legend Snippet: Interaction of RUNX2 with RUVBL2, INTS3 and BAZ1B. (A) The functional domains of RUNX2, RUVBL2, INTS3 and BAZ1B, and the location of peptide sequences identified by mass spectrometry. The peptide fragment identified by mass spectrometry is indicated as a closed bar. Functional domains in each peptide are indicated. RHD, Runt homolog domain; NMTS, nuclear matrix targeting sequences; AAA, ATPase associated with a variety of cellular activities; KD, kinase domain; DDT, DNA binding homeobox and different transcription factors; PHD, plant homeodomain; BRD, bromodomain. (B) Co-immunoprecipitation of RUNX2 with interacting proteins was analyzed by western blotting. To detect RUNX2–RUVBL2, RUNX2–INTS3 or RUNX2–BAZ1B endogenous interactions, 5 mg of whole-cell lysates from Saos2 or U2OS cells were immunoprecipitated (IP) with 5 µg of anti-RUNX2 antibodies or 5 µg of normal rabbit IgG as a negative control. Immunoprecipitation products were then analyzed by western blotting, using anti-RUVBL2, anti-INTS3 or anti-BAZ1B antibodies. Note that no clear immunoprecipitation products were seen using anti-INTS3 antibodies and the results are not shown. (C) Co-immunoprecipitation of FLAG–RUVBL2 protein with full-length RUNX2 [wildtype (WT), amino acids 1–528] or C-terminally deleted mutant (ΔC, amino acids 1–376). U2OS cells were transiently co-transfected with a FLAG–RUVBL2 expression construct and either full-length or C-terminally deleted RUNX2 construct. Whole-cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma). Washed beads were subjected to SDS-PAGE and analyzed by western blotting (WB) using specific antibodies against the indicated proteins. Asterisks (*) mark bands caused by nonspecific interactions. (D) Bacterially expressed GST (‘G’), GST fused to the Runt homolog domain of RUNX2 (amino acids 107–241; GST-R) or GST fused to the C-terminus of RUNX2 (amino acids 240–528; GST-C) proteins were immobilized on glutathione beads and incubated with whole-cell lysates from Saos2 cells. After extensive washing, proteins bound to the beads were eluted in protein sample buffer and analyzed by western blotting with antibodies against the indicated proteins.

Techniques Used: Functional Assay, Mass Spectrometry, Binding Assay, Immunoprecipitation, Western Blot, Negative Control, Mutagenesis, Transfection, Expressing, Construct, Incubation, SDS Page

8) Product Images from "NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2"

Article Title: NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2

Journal: EMBO Reports

doi: 10.15252/embr.201540505

NAT 10 interacts with p53 and Mdm2 U2 OS cells were transfected with Flag‐ NAT 10 or control vectors. Forty‐eight hours later, cells were harvested and whole‐cell extracts were immunoprecipitated with Flag antibody affinity resin. The NAT 10‐binding proteins were resolved by SDS – PAGE and detected by silver staining. U2 OS , HCT 116 p53 +/+ , or HCT 116 p53 −/− cell lysates were immunoprecipitated with control IgG, anti‐ NAT 10 (B and F), anti‐p53 (C), and anti‐Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. Purified NAT 10 was incubated with GST , GST ‐p53, or GST ‐Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by Western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel. Full‐length GST ‐p53 fusion protein, its deletion mutants, or GST protein was used in pull‐down experiments with purified NAT 10 protein. The levels of the GST fusion proteins are shown in the left panel. GST pull‐down assay was performed using purified GST ‐ NAT 10 deletion mutants or GST protein and overexpressed Flag‐p53 or Mdm2 protein in HEK 293T cells. Schematic diagram represents the constructs of GST ‐ NAT 10 deletion mutants (right panel).
Figure Legend Snippet: NAT 10 interacts with p53 and Mdm2 U2 OS cells were transfected with Flag‐ NAT 10 or control vectors. Forty‐eight hours later, cells were harvested and whole‐cell extracts were immunoprecipitated with Flag antibody affinity resin. The NAT 10‐binding proteins were resolved by SDS – PAGE and detected by silver staining. U2 OS , HCT 116 p53 +/+ , or HCT 116 p53 −/− cell lysates were immunoprecipitated with control IgG, anti‐ NAT 10 (B and F), anti‐p53 (C), and anti‐Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. Purified NAT 10 was incubated with GST , GST ‐p53, or GST ‐Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by Western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel. Full‐length GST ‐p53 fusion protein, its deletion mutants, or GST protein was used in pull‐down experiments with purified NAT 10 protein. The levels of the GST fusion proteins are shown in the left panel. GST pull‐down assay was performed using purified GST ‐ NAT 10 deletion mutants or GST protein and overexpressed Flag‐p53 or Mdm2 protein in HEK 293T cells. Schematic diagram represents the constructs of GST ‐ NAT 10 deletion mutants (right panel).

Techniques Used: Transfection, Immunoprecipitation, Binding Assay, SDS Page, Silver Staining, Purification, Incubation, Western Blot, Pull Down Assay, Construct

9) Product Images from "Activation of the Small G Protein Arf6 by Dynamin2 through Guanine Nucleotide Exchange Factors in Endocytosis"

Article Title: Activation of the Small G Protein Arf6 by Dynamin2 through Guanine Nucleotide Exchange Factors in Endocytosis

Journal: Scientific Reports

doi: 10.1038/srep14919

Dyn2 activates Arf6 in a manner dependent on its GTPase activity. ( A ) HA-tagged wild type of Dyn2 or its GTPase-deficient mutant K44A was coexpressed with Arf6-Flag in HeLa cells. After 24 hr, the active GTP-Arf6 was pulled down with glutathione-Sepharose beads conjugated with glutathione S -transferase (GST)-tagged leucine zipper region II (LZII) (amino acids 398–455) of JNK-interacting protein (JIP), which specifically binds to the active form of Arf6, and immunoblotted with anti-Flag antibody (left panel). Total Arf6 and Dyn2 expressed in the cell were also immunoblotted with anti-Flag and -HA antibodies, respectively. Right panel shows the means ± SEM of the levels of GTP-Arf6 from eight independent experiments. Statistical significance was calculated using Tukey multiple comparison test; ** P
Figure Legend Snippet: Dyn2 activates Arf6 in a manner dependent on its GTPase activity. ( A ) HA-tagged wild type of Dyn2 or its GTPase-deficient mutant K44A was coexpressed with Arf6-Flag in HeLa cells. After 24 hr, the active GTP-Arf6 was pulled down with glutathione-Sepharose beads conjugated with glutathione S -transferase (GST)-tagged leucine zipper region II (LZII) (amino acids 398–455) of JNK-interacting protein (JIP), which specifically binds to the active form of Arf6, and immunoblotted with anti-Flag antibody (left panel). Total Arf6 and Dyn2 expressed in the cell were also immunoblotted with anti-Flag and -HA antibodies, respectively. Right panel shows the means ± SEM of the levels of GTP-Arf6 from eight independent experiments. Statistical significance was calculated using Tukey multiple comparison test; ** P

Techniques Used: Activity Assay, Mutagenesis

10) Product Images from "Three SAUR proteins SAUR76, SAUR77 and SAUR78 promote plant growth in Arabidopsis"

Article Title: Three SAUR proteins SAUR76, SAUR77 and SAUR78 promote plant growth in Arabidopsis

Journal: Scientific Reports

doi: 10.1038/srep12477

Interaction of SAUR76-78 with ETR2 and their co-localization analysis. ( a ) Expressions of GST-SAUR fusion proteins. Arrows indicate positions of the corresponding GST-SAURs. GST was also noted as a degradation product. Numbers on the left indicate protein size markers. kD: kilodalton. ( b ) SAUR76-78 physically interact with ETR2 and EIN4 by GST pulldown. Upper panel: Each of the GST-SAURs can pulldown [ 35 S]-labeled ETR2 and EIN4. GST was used as a negative control. Lower panel: loading of the proteins by western analysis using anti-GST antibody. ( c ) Interaction of SAUR78 and SAUR76 with ETR2 by co-immunoprecipitation (Co-IP). Co-IP was performed with agarose beads conjugated with anti-Myc monoclonal antibody. The presence of the Flag-SAUR78, Flag-SAUR76 or Myc-ETR2 in the immunocomplex was detected with the anti-Flag or anti-Myc antibody by Western blotting. ( d ) Bimolecular fluorescence complementation (BiFC) assay. The Agrobacteria GV3101 haboring each of the two plasmids were co-infiltrated into tobacco leaves ( Nicotiana Benthamiana ). The samples were observed 48 h later under a confocal microscope. YFP fluorescence was excited at a wavelength of 488 nm. Bars indicate 25 μm. ( e ) Co-localization analysis of SAUR78 with ETR2. pGWB405-ETR2-GFP and pGWB454-SAUR78-RFP were transfected into Agrobacteria EHA105 and co-infiltrated into tobacco leaves. After infection for 3 d, fluorescence was observed under a confocal microscope. Bars indicate 25 μm.
Figure Legend Snippet: Interaction of SAUR76-78 with ETR2 and their co-localization analysis. ( a ) Expressions of GST-SAUR fusion proteins. Arrows indicate positions of the corresponding GST-SAURs. GST was also noted as a degradation product. Numbers on the left indicate protein size markers. kD: kilodalton. ( b ) SAUR76-78 physically interact with ETR2 and EIN4 by GST pulldown. Upper panel: Each of the GST-SAURs can pulldown [ 35 S]-labeled ETR2 and EIN4. GST was used as a negative control. Lower panel: loading of the proteins by western analysis using anti-GST antibody. ( c ) Interaction of SAUR78 and SAUR76 with ETR2 by co-immunoprecipitation (Co-IP). Co-IP was performed with agarose beads conjugated with anti-Myc monoclonal antibody. The presence of the Flag-SAUR78, Flag-SAUR76 or Myc-ETR2 in the immunocomplex was detected with the anti-Flag or anti-Myc antibody by Western blotting. ( d ) Bimolecular fluorescence complementation (BiFC) assay. The Agrobacteria GV3101 haboring each of the two plasmids were co-infiltrated into tobacco leaves ( Nicotiana Benthamiana ). The samples were observed 48 h later under a confocal microscope. YFP fluorescence was excited at a wavelength of 488 nm. Bars indicate 25 μm. ( e ) Co-localization analysis of SAUR78 with ETR2. pGWB405-ETR2-GFP and pGWB454-SAUR78-RFP were transfected into Agrobacteria EHA105 and co-infiltrated into tobacco leaves. After infection for 3 d, fluorescence was observed under a confocal microscope. Bars indicate 25 μm.

Techniques Used: Labeling, Negative Control, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Bimolecular Fluorescence Complementation Assay, Microscopy, Fluorescence, Transfection, Infection

11) Product Images from "Trs20 is Required for TRAPP II Assembly"

Article Title: Trs20 is Required for TRAPP II Assembly

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/tra.12065

TRAPP purified from trs20ts mutant cells does not contain TRAPP II and does not act as a Ypt32 GEF A. The protein level of Trs120-myc is significantly lower in purified TRAPP complexes, but not in lysates, from trs20ts when compared to wild type cells. GST-Bet5 and GST, as a negative control, were over-expressed in wild type (NSY1471) and trs20ts (NSY1472) mutant cells also expressing endogenously tagged Trs120-myc. Cells grown to mid-log phase were either left at 26° or shifted to 37° for 70 minutes and then harvested. Cell lysates were prepared and GST-Bet5 complexes were purified on glutathione sepharose resin. The level of Trs120-myc was determined in lysates (top) and pull-downs (bottom) using immuno-blot analysis; G6PDH level was used as a loading control for lysates; GST-Bet5 and GST levels are used for the pull down yield. B. The protein level of Trs130-HA is significantly lower in lysates and TRAPP complexes purified from trs20ts when compared to wild type cells. Same as in panel A, except that cells were expressing endogenously tagged Trs130-HA, and the pull-down of the TRAPP I/II subunit Bet3 was verified using anti-Bet3 antibodies. The partial degradation of over-expressed GST-Bet5 in trs20ts mutant cells in likely due to the instability of TRAPP complexes in these cells. For panels A and B, the level of Trs120-myc or Trs130-HA was quantified and shown under the immuno-blots as percent of wild type cells. Protein level in lysates was corrected for the loading control, while in pull downs it was corrected for the full-length GST-Bet5; +/− represents SEM; P values are shown on the right (values
Figure Legend Snippet: TRAPP purified from trs20ts mutant cells does not contain TRAPP II and does not act as a Ypt32 GEF A. The protein level of Trs120-myc is significantly lower in purified TRAPP complexes, but not in lysates, from trs20ts when compared to wild type cells. GST-Bet5 and GST, as a negative control, were over-expressed in wild type (NSY1471) and trs20ts (NSY1472) mutant cells also expressing endogenously tagged Trs120-myc. Cells grown to mid-log phase were either left at 26° or shifted to 37° for 70 minutes and then harvested. Cell lysates were prepared and GST-Bet5 complexes were purified on glutathione sepharose resin. The level of Trs120-myc was determined in lysates (top) and pull-downs (bottom) using immuno-blot analysis; G6PDH level was used as a loading control for lysates; GST-Bet5 and GST levels are used for the pull down yield. B. The protein level of Trs130-HA is significantly lower in lysates and TRAPP complexes purified from trs20ts when compared to wild type cells. Same as in panel A, except that cells were expressing endogenously tagged Trs130-HA, and the pull-down of the TRAPP I/II subunit Bet3 was verified using anti-Bet3 antibodies. The partial degradation of over-expressed GST-Bet5 in trs20ts mutant cells in likely due to the instability of TRAPP complexes in these cells. For panels A and B, the level of Trs120-myc or Trs130-HA was quantified and shown under the immuno-blots as percent of wild type cells. Protein level in lysates was corrected for the loading control, while in pull downs it was corrected for the full-length GST-Bet5; +/− represents SEM; P values are shown on the right (values

Techniques Used: Purification, Mutagenesis, Activated Clotting Time Assay, Negative Control, Expressing, Western Blot

Trs20 is required for interaction of Trs120 with recombinant TRAPP I A. Pull down of His 6 -Trs120 with GST-Bet5 purified complexes. GST was pulled down, using glutathione sepharose resin, from lysates of bacteria expressing core TRAPP I (GST-Bet5, Trs23-S, Trs31-myc, and Bet3-MBP), core TRAPP I plus Trs33 (His 6 -Trs33), core TRAPP I plus Trs20 (Trs20-HA), or core TRAPP I plus Trs20 and Trs33. Cleared lysate (S100) from different bacterial cells expressing His 6 -Trs120 was then incubated with the resin and the level of proteins associated with the resin after precipitation was determined using immuno-blot analysis and antibodies against the tags (Trs120, Bet5, Trs33, Trs20) or the protein (Bet3). Trs120 co-purifies with GST-Bet5 complex ( > 5%), but not with GST, and only in the presence of Trs20. More Trs120 co-purifies with TRAPP when Trs33 is present ( > 10%). The expression levels of the different proteins in lysates are shown on the left (10% input for the Trs120 lysate). The full anti-His 6 . B. Pull down of His 6 -Trs120 with Bet3-MBP purified complexes. Binding of His 6 -Trs120 TRAPP I was determined as in part A (using the same lysates), except that amylose resin was used to pull down Bet3-MBP within the core TRAPP I complex. Trs120 co-purifies with TRAPP I only in the presence of Trs20 ( > 5%), and this level is higher in the presence of Trs33 ( > 10%). C. Pull down of core TRAPP I with His 6 -Trs120. Lysates from bacteria expressing His 6 -Trs120, or empty plasmid (θ) as a negative control, were purified on Ni 2+ resin. The resin was then incubated with lysates from cells expressing either core TRAPP I, or core TRAPP I plus Trs20-HA. The level of proteins associated with the resin after precipitation was determined using immuno-blot analysis and antibodies against the tags. TRAPP I, scored by the level of Trs31-myc, co-purified with Trs120, but not with the empty plasmid control, and only in the presence of Trs20. In A–C, asterisks indicate the tagged protein being bound directly to the resin. Results in this figure are representative of at least two independent experiments.
Figure Legend Snippet: Trs20 is required for interaction of Trs120 with recombinant TRAPP I A. Pull down of His 6 -Trs120 with GST-Bet5 purified complexes. GST was pulled down, using glutathione sepharose resin, from lysates of bacteria expressing core TRAPP I (GST-Bet5, Trs23-S, Trs31-myc, and Bet3-MBP), core TRAPP I plus Trs33 (His 6 -Trs33), core TRAPP I plus Trs20 (Trs20-HA), or core TRAPP I plus Trs20 and Trs33. Cleared lysate (S100) from different bacterial cells expressing His 6 -Trs120 was then incubated with the resin and the level of proteins associated with the resin after precipitation was determined using immuno-blot analysis and antibodies against the tags (Trs120, Bet5, Trs33, Trs20) or the protein (Bet3). Trs120 co-purifies with GST-Bet5 complex ( > 5%), but not with GST, and only in the presence of Trs20. More Trs120 co-purifies with TRAPP when Trs33 is present ( > 10%). The expression levels of the different proteins in lysates are shown on the left (10% input for the Trs120 lysate). The full anti-His 6 . B. Pull down of His 6 -Trs120 with Bet3-MBP purified complexes. Binding of His 6 -Trs120 TRAPP I was determined as in part A (using the same lysates), except that amylose resin was used to pull down Bet3-MBP within the core TRAPP I complex. Trs120 co-purifies with TRAPP I only in the presence of Trs20 ( > 5%), and this level is higher in the presence of Trs33 ( > 10%). C. Pull down of core TRAPP I with His 6 -Trs120. Lysates from bacteria expressing His 6 -Trs120, or empty plasmid (θ) as a negative control, were purified on Ni 2+ resin. The resin was then incubated with lysates from cells expressing either core TRAPP I, or core TRAPP I plus Trs20-HA. The level of proteins associated with the resin after precipitation was determined using immuno-blot analysis and antibodies against the tags. TRAPP I, scored by the level of Trs31-myc, co-purified with Trs120, but not with the empty plasmid control, and only in the presence of Trs20. In A–C, asterisks indicate the tagged protein being bound directly to the resin. Results in this figure are representative of at least two independent experiments.

Techniques Used: Recombinant, Purification, Expressing, Incubation, Binding Assay, Plasmid Preparation, Negative Control

12) Product Images from "The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿"

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01135-09

Xic1 interacts with DDB1 and XCdt2. (A) Schematic representation of XCdt2. XCdt2 contains two WDXR motifs (gray boxes), six WD40 domains (white boxes), and a conserved arginine residue (R247) essential for DDB1 binding. (B) GST pulldown assay. GST or GST-Xic1 immobilized on glutathione-Sepharose beads was incubated with Xenopus interphase egg extract and immunoblotted with antibody against Xenopus DDB1 and PCNA (Western blot). GST and GST-Xic1 proteins (20% of Western blot reaction) were stained with Coomassie brilliant blue. The input (4%) is shown in lane 1. α, anti; *, nonspecific bacterial protein. (C) Coimmunoprecipitation assay. Immunoprecipitated DDB1 (IP) from the egg extract was bound to protein A beads and incubated with 35 S-labeled wild-type XCdt2 (WT), XCdt2 R247A (R247A), XCdt2 1-400 (1-400), or XCdt2 401-710 (401-710). As a control, nonspecific normal rabbit serum (NRS) was used in the place of DDB1 antiserum. Efficient immunoprecipitation of XDDB1 was confirmed by immunoblotting with anti-DDB1 antibody (top). Binding of XCdt2 proteins ( 35 S-Cdt2) was analyzed by SDS-PAGE and phosphorimaging, and 5% of the input proteins is shown (5% input). (D) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with 35 S-labeled Xic1 and subjected to SDS-PAGE and phosphorimager analysis. Efficient immunoprecipitation of XCdt2 was confirmed by immunoblotting with anti-Cdt2 antibody (top). Immunoprecipitation with normal rabbit serum (NRS) was included as a control, and input samples are indicated. (E) GST pulldown assay. Bacterially expressed GST or GST-Xic1 (5 μg) was immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled XCdt2 proteins, as indicated. A total of 5% of the input reaction is shown. The percentage of Cdt2 bound by all GST-Xic1 proteins (% binding) is an average value obtained from 2 independent experiments.
Figure Legend Snippet: Xic1 interacts with DDB1 and XCdt2. (A) Schematic representation of XCdt2. XCdt2 contains two WDXR motifs (gray boxes), six WD40 domains (white boxes), and a conserved arginine residue (R247) essential for DDB1 binding. (B) GST pulldown assay. GST or GST-Xic1 immobilized on glutathione-Sepharose beads was incubated with Xenopus interphase egg extract and immunoblotted with antibody against Xenopus DDB1 and PCNA (Western blot). GST and GST-Xic1 proteins (20% of Western blot reaction) were stained with Coomassie brilliant blue. The input (4%) is shown in lane 1. α, anti; *, nonspecific bacterial protein. (C) Coimmunoprecipitation assay. Immunoprecipitated DDB1 (IP) from the egg extract was bound to protein A beads and incubated with 35 S-labeled wild-type XCdt2 (WT), XCdt2 R247A (R247A), XCdt2 1-400 (1-400), or XCdt2 401-710 (401-710). As a control, nonspecific normal rabbit serum (NRS) was used in the place of DDB1 antiserum. Efficient immunoprecipitation of XDDB1 was confirmed by immunoblotting with anti-DDB1 antibody (top). Binding of XCdt2 proteins ( 35 S-Cdt2) was analyzed by SDS-PAGE and phosphorimaging, and 5% of the input proteins is shown (5% input). (D) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with 35 S-labeled Xic1 and subjected to SDS-PAGE and phosphorimager analysis. Efficient immunoprecipitation of XCdt2 was confirmed by immunoblotting with anti-Cdt2 antibody (top). Immunoprecipitation with normal rabbit serum (NRS) was included as a control, and input samples are indicated. (E) GST pulldown assay. Bacterially expressed GST or GST-Xic1 (5 μg) was immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled XCdt2 proteins, as indicated. A total of 5% of the input reaction is shown. The percentage of Cdt2 bound by all GST-Xic1 proteins (% binding) is an average value obtained from 2 independent experiments.

Techniques Used: Binding Assay, GST Pulldown Assay, Incubation, Western Blot, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Labeling, SDS Page

Xic1 turnover does not require the tandem arrangement of PCNA and Cdt2 binding domains. (A) Amino acid sequence alignment of p21 (p21Cip1) and Xic1 (p27Xic1). Cdt2 binding regions indicated by italicized amino acid residues and bold lines, the PCNA binding element (PIP box) indicated by gray box, and critical lysine residues of Xic1 indicated by underlining, italicizing, and boldfacing of amino acid residues. (B) Schematic representation of mutant Xic1 proteins. CDK2-cyclin and wild-type PCNA binding domains are indicated by dark gray shading, while the I174A PCNA binding mutant is indicated by a white box. Xic1 residue numbers are indicated below each schematic. The NPIP1 and NPIP2 domains are fused to the N terminus of wild-type Xic1 (WT-NPIP), Xic1-I174A (I174A-NPIP), or amino acids 1 to 160 of Xic1 (N160-NPIP) as indicated and includes Xic1 amino acids 171 to 186 (TTPITDYFPKRKKILS) for NPIP1 and p21 residues 135 to 164 with an internal deletion of residues 156 to 161 for NPIP2. The NPIP2 domain serves solely as a PCNA binding domain and does not retain the ability to efficiently bind Cdt2. (C) GST pulldown assay. GST or GST-Xic1 wild-type and mutant proteins (top, NPIP1; bottom, NPIP2) were immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled Xenopus Cdt2 ( 35 S-XCDT2). The 35 S-XCdt2 input control (5% input) is shown in lane 1. (D) Xic1 degradation assay. (Top and middle) 35 S-labeled Xic1 wild-type (WT) and mutant proteins (WT-NPIP2, I174A, I174A-NPIP2, and N160-NPIP2) as indicated were incubated in HSS with (+) or without (−) single-stranded DNA for the indicated times, followed by SDS-PAGE and phosphorimager analysis. Asterisks indicate internal initiation translation products. (Bottom) Quantitation of Xic1 degradation. The mean percentage of Xic1 remaining from two (WT, WT-NPIP1, I174A-NPIP1, and N160-NPIP1) or three (WT-NPIP2, I174A, I174A-NPIP2, and N160-NPIP2) independent experiments as described above is shown, where the 0-h time point was normalized to 100% of Xic1 remaining for each sample. SEMs are shown as error bars. (E) Quantitation of Xic1 binding to PCNA. GST or GST-PCNA proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled Xic1 wild-type (WT) or mutant proteins (I174A, WT-NPIP1, I174A-NPIP1, N160-NPIP1, WT-NPIP2, I174A-NPIP2, and N160-NPIP2). The average percentage of Xic1 bound by GST-PCNA (% PCNA binding) is shown, where values for WT Xic1 and I174A are averages of results from 4 independent experiments, and the values of the NPIP mutants (WT-NPIP1, I174A-NPIP1, N160-NPIP1, WT-NPIP2, I174A-NPIP2, and N160-NPIP2) are averages of results from 2 independent experiments. SEMs are shown as error bars.
Figure Legend Snippet: Xic1 turnover does not require the tandem arrangement of PCNA and Cdt2 binding domains. (A) Amino acid sequence alignment of p21 (p21Cip1) and Xic1 (p27Xic1). Cdt2 binding regions indicated by italicized amino acid residues and bold lines, the PCNA binding element (PIP box) indicated by gray box, and critical lysine residues of Xic1 indicated by underlining, italicizing, and boldfacing of amino acid residues. (B) Schematic representation of mutant Xic1 proteins. CDK2-cyclin and wild-type PCNA binding domains are indicated by dark gray shading, while the I174A PCNA binding mutant is indicated by a white box. Xic1 residue numbers are indicated below each schematic. The NPIP1 and NPIP2 domains are fused to the N terminus of wild-type Xic1 (WT-NPIP), Xic1-I174A (I174A-NPIP), or amino acids 1 to 160 of Xic1 (N160-NPIP) as indicated and includes Xic1 amino acids 171 to 186 (TTPITDYFPKRKKILS) for NPIP1 and p21 residues 135 to 164 with an internal deletion of residues 156 to 161 for NPIP2. The NPIP2 domain serves solely as a PCNA binding domain and does not retain the ability to efficiently bind Cdt2. (C) GST pulldown assay. GST or GST-Xic1 wild-type and mutant proteins (top, NPIP1; bottom, NPIP2) were immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled Xenopus Cdt2 ( 35 S-XCDT2). The 35 S-XCdt2 input control (5% input) is shown in lane 1. (D) Xic1 degradation assay. (Top and middle) 35 S-labeled Xic1 wild-type (WT) and mutant proteins (WT-NPIP2, I174A, I174A-NPIP2, and N160-NPIP2) as indicated were incubated in HSS with (+) or without (−) single-stranded DNA for the indicated times, followed by SDS-PAGE and phosphorimager analysis. Asterisks indicate internal initiation translation products. (Bottom) Quantitation of Xic1 degradation. The mean percentage of Xic1 remaining from two (WT, WT-NPIP1, I174A-NPIP1, and N160-NPIP1) or three (WT-NPIP2, I174A, I174A-NPIP2, and N160-NPIP2) independent experiments as described above is shown, where the 0-h time point was normalized to 100% of Xic1 remaining for each sample. SEMs are shown as error bars. (E) Quantitation of Xic1 binding to PCNA. GST or GST-PCNA proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled Xic1 wild-type (WT) or mutant proteins (I174A, WT-NPIP1, I174A-NPIP1, N160-NPIP1, WT-NPIP2, I174A-NPIP2, and N160-NPIP2). The average percentage of Xic1 bound by GST-PCNA (% PCNA binding) is shown, where values for WT Xic1 and I174A are averages of results from 4 independent experiments, and the values of the NPIP mutants (WT-NPIP1, I174A-NPIP1, N160-NPIP1, WT-NPIP2, I174A-NPIP2, and N160-NPIP2) are averages of results from 2 independent experiments. SEMs are shown as error bars.

Techniques Used: Binding Assay, Sequencing, Mutagenesis, GST Pulldown Assay, Incubation, Labeling, Degradation Assay, SDS Page, Quantitation Assay

PCNA directly interacts with the C-terminal domain of XCdt2. (A) GST pulldown assay. Bacterially expressed GST, GST-XCdt2 1-400 , or GST-XCdt2 401-710 was bound to glutathione-Sepharose and incubated with purified XPCNA (0, 5, 25, and 50 μg) or bovine serum albumin (BSA; 0 and 50 μg) (left) as indicated and MBP-Xic1 (0, 5, 25, and 50 μg) (right), followed by staining with Coomassie blue. Protein bands were identified by mass spectrometry and are labeled accordingly. Several bacterial contaminants were identified. “+” was identified as the bacterial DnaK protein, and “*” was identified as the bacterial GroEL protein. (B) GST pulldown and competitive binding assay. Bacterially expressed GST or GST-PCNA (5 μg) was bound to glutathione-Sepharose beads and incubated with 0 to 50 μg of purified MBP-Xic1 or GST as indicated and 35 S-labeled wild-type XCdt2. (C) GST pulldown and competition study. GST or GST-PCNA was bound to glutathione-Sepharose beads and incubated with 0 to 50 μg of purified MBP-Xic1 as indicated. Following a washing step, samples were incubated with 35 S-labeled XCdt2. (D) GST pulldown assay and competitive binding assay. GST, GST-Xic1 WT , or GST-Xic1 I174A bound to glutathione-Sepharose beads was incubated with 0 to 50 μg of purified XPCNA and 35 S-labeled wild-type XCdt2. (B to D) Samples were analyzed by Coomassie blue staining and phosphorimaging. (Left) Schematic representation of proteins analyzed in binding assays. (Right) The average relative Cdt2 binding values [relative Cdt2 binding (%)] of results from at least 2 independent experiments are shown, where the “zero competitor” value was normalized to 100%.
Figure Legend Snippet: PCNA directly interacts with the C-terminal domain of XCdt2. (A) GST pulldown assay. Bacterially expressed GST, GST-XCdt2 1-400 , or GST-XCdt2 401-710 was bound to glutathione-Sepharose and incubated with purified XPCNA (0, 5, 25, and 50 μg) or bovine serum albumin (BSA; 0 and 50 μg) (left) as indicated and MBP-Xic1 (0, 5, 25, and 50 μg) (right), followed by staining with Coomassie blue. Protein bands were identified by mass spectrometry and are labeled accordingly. Several bacterial contaminants were identified. “+” was identified as the bacterial DnaK protein, and “*” was identified as the bacterial GroEL protein. (B) GST pulldown and competitive binding assay. Bacterially expressed GST or GST-PCNA (5 μg) was bound to glutathione-Sepharose beads and incubated with 0 to 50 μg of purified MBP-Xic1 or GST as indicated and 35 S-labeled wild-type XCdt2. (C) GST pulldown and competition study. GST or GST-PCNA was bound to glutathione-Sepharose beads and incubated with 0 to 50 μg of purified MBP-Xic1 as indicated. Following a washing step, samples were incubated with 35 S-labeled XCdt2. (D) GST pulldown assay and competitive binding assay. GST, GST-Xic1 WT , or GST-Xic1 I174A bound to glutathione-Sepharose beads was incubated with 0 to 50 μg of purified XPCNA and 35 S-labeled wild-type XCdt2. (B to D) Samples were analyzed by Coomassie blue staining and phosphorimaging. (Left) Schematic representation of proteins analyzed in binding assays. (Right) The average relative Cdt2 binding values [relative Cdt2 binding (%)] of results from at least 2 independent experiments are shown, where the “zero competitor” value was normalized to 100%.

Techniques Used: GST Pulldown Assay, Incubation, Purification, Staining, Mass Spectrometry, Labeling, Competitive Binding Assay, Binding Assay

p21 is ubiquitinated during the events of DNA polymerase switching/elongation in the Xenopus egg extract. (A) Amino acid sequence similarity between Xenopus and human Cul4a, Cul4b, DDB1, Cdt2, and PCNA. Xenopus residue numbers are indicated at the bottom of the sequence alignments, and the percentages of similarity (S) and identity (I) between the Xenopus and human proteins are shown on the right. Xenopus Cul4a, the MGC115611 protein (gi 71679818), contains 200 additional residues in the N terminus compared to human Cul4a, so only residues 200 to 858 of Xenopus Cul4a were compared in the alignment. (B) GST pulldown assay. GST, GST-p21, or GST-p27 proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-hCDT2. A total of 5% of the input hCdt2 is shown (5% input). (C) Schematic representation of p21 mutants. CDK-cyclin and PCNA binding domains for untagged and GST-tagged p21 mutants are indicated. In the p21 point mutant F150A, phenylalanine is replaced by alanine at residue 150. Mutant Δ156-161 contains a deletion of residues 156 to 161, while other deletion mutants are named by the remaining residues of p21. (D) GST pulldown assay. (Top) GST or GST-p21 wild-type or mutant proteins were bound to glutathione-Sepharose beads, followed by incubation with 10 μl of HSS in NETN buffer. The bead fraction was analyzed by immunoblotting with anti-hPCNA antibody (Santa Cruz), and 0.5 μl HSS was included as an input control (5% input). (Bottom) GST or GST-p21 wild-type or mutant proteins were immobilized onto glutathione-Sepharose beads, followed by incubation with 35 S-hCDT2 and analysis by SDS-PAGE and phosphorimaging. The average percentage of hCdt2 bound (ave % CDT2 binding) was calculated using results from 2 independent experiments and was normalized to the level of hCdt2 binding to wild-type p21, which was set at 100%. (E) p21 ubiquitination and degradation assay. 35 S-labeled wild-type p21 was incubated in HSS supplemented with 2.5 μl XB − buffer, unprogrammed reticulocyte lysate (unprog; lysate programmed with vector DNA), or in vitro -translated hCdt2 with (+) or without (−) single-stranded DNA (ssDNA). Samples were analyzed at time points between 0 and 180 min as indicated. Ubiquitinated p21 species (Ub n ) are shown on the right, and molecular mass markers are shown in kilodaltons on the left. The percentage of p21 remaining at each time point was calculated as a percentage of the amount of p21 at the zero time point, which was normalized to 100%. (F) p21 ubiquitination assay. 35 S-labeled wild-type p21 (WT), the p21 F150A point mutant (F150A), or the p21 Δ156-161 deletion mutant (Δ156-161) was incubated in HSS supplemented with 2.5 μl in vitro -translated Xenopus Cdt2 (XCdt2) or human Cdt2 (hCdt2) as indicated in the presence (+) or absence (−) of single-stranded DNA (ssDNA), followed by analysis at 0 and 120 min. Ubiquitinated p21 species (Ub n ) are shown on the left, and molecular mass markers are shown in kilodaltons on the right.
Figure Legend Snippet: p21 is ubiquitinated during the events of DNA polymerase switching/elongation in the Xenopus egg extract. (A) Amino acid sequence similarity between Xenopus and human Cul4a, Cul4b, DDB1, Cdt2, and PCNA. Xenopus residue numbers are indicated at the bottom of the sequence alignments, and the percentages of similarity (S) and identity (I) between the Xenopus and human proteins are shown on the right. Xenopus Cul4a, the MGC115611 protein (gi 71679818), contains 200 additional residues in the N terminus compared to human Cul4a, so only residues 200 to 858 of Xenopus Cul4a were compared in the alignment. (B) GST pulldown assay. GST, GST-p21, or GST-p27 proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-hCDT2. A total of 5% of the input hCdt2 is shown (5% input). (C) Schematic representation of p21 mutants. CDK-cyclin and PCNA binding domains for untagged and GST-tagged p21 mutants are indicated. In the p21 point mutant F150A, phenylalanine is replaced by alanine at residue 150. Mutant Δ156-161 contains a deletion of residues 156 to 161, while other deletion mutants are named by the remaining residues of p21. (D) GST pulldown assay. (Top) GST or GST-p21 wild-type or mutant proteins were bound to glutathione-Sepharose beads, followed by incubation with 10 μl of HSS in NETN buffer. The bead fraction was analyzed by immunoblotting with anti-hPCNA antibody (Santa Cruz), and 0.5 μl HSS was included as an input control (5% input). (Bottom) GST or GST-p21 wild-type or mutant proteins were immobilized onto glutathione-Sepharose beads, followed by incubation with 35 S-hCDT2 and analysis by SDS-PAGE and phosphorimaging. The average percentage of hCdt2 bound (ave % CDT2 binding) was calculated using results from 2 independent experiments and was normalized to the level of hCdt2 binding to wild-type p21, which was set at 100%. (E) p21 ubiquitination and degradation assay. 35 S-labeled wild-type p21 was incubated in HSS supplemented with 2.5 μl XB − buffer, unprogrammed reticulocyte lysate (unprog; lysate programmed with vector DNA), or in vitro -translated hCdt2 with (+) or without (−) single-stranded DNA (ssDNA). Samples were analyzed at time points between 0 and 180 min as indicated. Ubiquitinated p21 species (Ub n ) are shown on the right, and molecular mass markers are shown in kilodaltons on the left. The percentage of p21 remaining at each time point was calculated as a percentage of the amount of p21 at the zero time point, which was normalized to 100%. (F) p21 ubiquitination assay. 35 S-labeled wild-type p21 (WT), the p21 F150A point mutant (F150A), or the p21 Δ156-161 deletion mutant (Δ156-161) was incubated in HSS supplemented with 2.5 μl in vitro -translated Xenopus Cdt2 (XCdt2) or human Cdt2 (hCdt2) as indicated in the presence (+) or absence (−) of single-stranded DNA (ssDNA), followed by analysis at 0 and 120 min. Ubiquitinated p21 species (Ub n ) are shown on the left, and molecular mass markers are shown in kilodaltons on the right.

Techniques Used: Sequencing, GST Pulldown Assay, Incubation, Binding Assay, Mutagenesis, SDS Page, Degradation Assay, Labeling, Plasmid Preparation, In Vitro, Ubiquitin Assay

Xic1 residues immediately upstream and downstream of its PCNA binding domain are important for Cdt2 binding. (A) Schematic representation of full-length Xic1 and Xic1 deletion mutants, with CDK/cyclin and PCNA binding domains indicated. Amino- or carboxy-terminal serial deletion mutants of Xic1 were in vitro -translated ( 35 S-Xic1) or bacterially expressed as GST-Xic1 fusion proteins (GST-Xic1). The Xic1 wild type (WT), point mutant I174A deficient for PCNA binding (I174A), CK − mutant deficient for CDK2-cyclin binding (CK − ), or Xic1 deletion mutants indicated by the residues contained within the mutant or deleted (Δ) within the mutant are shown. (B) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with the 35 S-Xic1 wild type (WT) or mutants as indicated. Equivalent immunoprecipitation of XCdt2 for each sample was confirmed by immunoblotting with anti-Cdt2 antibody (data not shown). Immunoprecipitation with normal rabbit serum (NRS) was conducted as a control, and 5% of the input 35 S-Xic1 is shown (5% input). (C) GST pulldown assay. GST or GST-Xic1 wild-type or mutant proteins as indicated were immobilized on glutathione-Sepharose beads and incubated with 35 S-CDT2. A total of 5% of the input XCdt2 for each reaction is shown (5% input). (D) Quantitation of the results shown in panels B and C. The relative XCdt2 binding value (% relative Cdt2 binding) for each Xic1 mutant is shown, where wild-type Xic1 (WT) binding was normalized to 100% for each experiment. Each sample was tested at least 2 or 3 times, and the standard error of the mean (SEM) is shown as an error bar for samples tested at least three times. IVT, in vitro transcribed.
Figure Legend Snippet: Xic1 residues immediately upstream and downstream of its PCNA binding domain are important for Cdt2 binding. (A) Schematic representation of full-length Xic1 and Xic1 deletion mutants, with CDK/cyclin and PCNA binding domains indicated. Amino- or carboxy-terminal serial deletion mutants of Xic1 were in vitro -translated ( 35 S-Xic1) or bacterially expressed as GST-Xic1 fusion proteins (GST-Xic1). The Xic1 wild type (WT), point mutant I174A deficient for PCNA binding (I174A), CK − mutant deficient for CDK2-cyclin binding (CK − ), or Xic1 deletion mutants indicated by the residues contained within the mutant or deleted (Δ) within the mutant are shown. (B) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with the 35 S-Xic1 wild type (WT) or mutants as indicated. Equivalent immunoprecipitation of XCdt2 for each sample was confirmed by immunoblotting with anti-Cdt2 antibody (data not shown). Immunoprecipitation with normal rabbit serum (NRS) was conducted as a control, and 5% of the input 35 S-Xic1 is shown (5% input). (C) GST pulldown assay. GST or GST-Xic1 wild-type or mutant proteins as indicated were immobilized on glutathione-Sepharose beads and incubated with 35 S-CDT2. A total of 5% of the input XCdt2 for each reaction is shown (5% input). (D) Quantitation of the results shown in panels B and C. The relative XCdt2 binding value (% relative Cdt2 binding) for each Xic1 mutant is shown, where wild-type Xic1 (WT) binding was normalized to 100% for each experiment. Each sample was tested at least 2 or 3 times, and the standard error of the mean (SEM) is shown as an error bar for samples tested at least three times. IVT, in vitro transcribed.

Techniques Used: Binding Assay, In Vitro, Mutagenesis, Co-Immunoprecipitation Assay, Immunoprecipitation, Incubation, GST Pulldown Assay, Quantitation Assay

13) Product Images from "The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases"

Article Title: The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201705068

TRAPPII complex activates the Rab GTPases Rab1 and Rab11, whereas TRAPPIII only shows GEF activity toward Rab1. (A) Coomassie blue–stained protein gels of recombinant Drosophila TRAPP complexes purified from Sf9 cells coexpressing the subunits of each complex. FLAG tags on C10 or C11 allowed isolation of TRAPPII or TRAPPIII, respectively. PreScission protease (GST-HRV-3C protease) was used to cleave the tags (asterisks) and was subsequently removed using glutathione Sepharose beads. C10 also copurified with C9 in the absence of the shared subunits (TRAPPII lane). The Hsc70 chaperone (CG4264) is a contaminant of the TRAPPII purification protocol. Molecular masses are given in kilodaltons. (B) Release of mant-GDP from 250 nM of Rab-His 6 by 50 nM of TRAPPII or TRAPPIII in the presence or absence of synthetic fly Golgi mix liposomes. Traces are the mean of at least three experiments. Error bars show SEM.
Figure Legend Snippet: TRAPPII complex activates the Rab GTPases Rab1 and Rab11, whereas TRAPPIII only shows GEF activity toward Rab1. (A) Coomassie blue–stained protein gels of recombinant Drosophila TRAPP complexes purified from Sf9 cells coexpressing the subunits of each complex. FLAG tags on C10 or C11 allowed isolation of TRAPPII or TRAPPIII, respectively. PreScission protease (GST-HRV-3C protease) was used to cleave the tags (asterisks) and was subsequently removed using glutathione Sepharose beads. C10 also copurified with C9 in the absence of the shared subunits (TRAPPII lane). The Hsc70 chaperone (CG4264) is a contaminant of the TRAPPII purification protocol. Molecular masses are given in kilodaltons. (B) Release of mant-GDP from 250 nM of Rab-His 6 by 50 nM of TRAPPII or TRAPPIII in the presence or absence of synthetic fly Golgi mix liposomes. Traces are the mean of at least three experiments. Error bars show SEM.

Techniques Used: Activity Assay, Staining, Recombinant, Purification, Isolation

14) Product Images from "Antagonistic Interplay between Necdin and Bmi1 Controls Proliferation of Neural Precursor Cells in the Embryonic Mouse Neocortex"

Article Title: Antagonistic Interplay between Necdin and Bmi1 Controls Proliferation of Neural Precursor Cells in the Embryonic Mouse Neocortex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084460

Necdin interacts with Bmi1 in vivo and in vitro . ( A ) Bmi1 deletion mutants. Bmi1 full-length (FL), C-terminal deletion (ΔCT), and mutants containing the Ring finger (RF), helix-turn-helix (HTH) and proline/serine-rich (PS) domains are schematically shown. ( B ) Co-immunoprecipitation assay. HEK293A cells were transfected with expression vectors for necdin and Myc-tagged FL, RF, HTH, p53 (positive control), and p53ΔN (negative control). Cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with anti-Myc (Myc) and anti-necdin (Necdin) antibodies. ( C ) In vitro binding assay. GST-Bmi1 mutants immobilized on glutathione-agarose were incubated with His-tagged necdin (His-necdin), and bound His-necdin was detected by immunoblotting with anti-necdin antibody (upper panel). GST-Bmi1 deletion mutants were stained with Coomassie Brilliant Blue (lower panel). Arrows indicate the predicted protein positions ( B , C ). ( D ) Co-immunoprecipitation assay for endogenous complex containing necdin and Bmi1 in primary NPCs. Lysates of NPCs prepared from E14.5 wild-type (WT) and necdin-null (KO) mice were immunoprecipitated with anti-necdin IgG (Nec) or control preimmune IgG (Pre). Bmi1, PCNA (negative control), and necdin were detected by Western blotting. Lysate, tissue lysate (10 µg).
Figure Legend Snippet: Necdin interacts with Bmi1 in vivo and in vitro . ( A ) Bmi1 deletion mutants. Bmi1 full-length (FL), C-terminal deletion (ΔCT), and mutants containing the Ring finger (RF), helix-turn-helix (HTH) and proline/serine-rich (PS) domains are schematically shown. ( B ) Co-immunoprecipitation assay. HEK293A cells were transfected with expression vectors for necdin and Myc-tagged FL, RF, HTH, p53 (positive control), and p53ΔN (negative control). Cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with anti-Myc (Myc) and anti-necdin (Necdin) antibodies. ( C ) In vitro binding assay. GST-Bmi1 mutants immobilized on glutathione-agarose were incubated with His-tagged necdin (His-necdin), and bound His-necdin was detected by immunoblotting with anti-necdin antibody (upper panel). GST-Bmi1 deletion mutants were stained with Coomassie Brilliant Blue (lower panel). Arrows indicate the predicted protein positions ( B , C ). ( D ) Co-immunoprecipitation assay for endogenous complex containing necdin and Bmi1 in primary NPCs. Lysates of NPCs prepared from E14.5 wild-type (WT) and necdin-null (KO) mice were immunoprecipitated with anti-necdin IgG (Nec) or control preimmune IgG (Pre). Bmi1, PCNA (negative control), and necdin were detected by Western blotting. Lysate, tissue lysate (10 µg).

Techniques Used: In Vivo, In Vitro, Co-Immunoprecipitation Assay, Transfection, Expressing, Positive Control, Negative Control, Immunoprecipitation, Binding Assay, Incubation, Staining, Mouse Assay, Western Blot

15) Product Images from "Intrasteric control of AMPK via the ?1 subunit AMP allosteric regulatory site"

Article Title: Intrasteric control of AMPK via the ?1 subunit AMP allosteric regulatory site

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.03340004

Effect of Arg mutations on AMP activation. Wild-type γ 1 or mutant γ 1 was cotransfected with α 1 and β 1 in COS-7 cells and AMPK αβγ holoenzyme was purified by Glutathione Sepharose chromatograph. AMPK activity was determined using the SAMS peptide (see Materials and Methods). The activity measured in the absence of AMP was subtracted from plus AMP values of the wild-type and Phe mutants and fitted to the one-site binding Michaelis-Menten Equation curve. The activity of the Arg mutants was AMP-independent.
Figure Legend Snippet: Effect of Arg mutations on AMP activation. Wild-type γ 1 or mutant γ 1 was cotransfected with α 1 and β 1 in COS-7 cells and AMPK αβγ holoenzyme was purified by Glutathione Sepharose chromatograph. AMPK activity was determined using the SAMS peptide (see Materials and Methods). The activity measured in the absence of AMP was subtracted from plus AMP values of the wild-type and Phe mutants and fitted to the one-site binding Michaelis-Menten Equation curve. The activity of the Arg mutants was AMP-independent.

Techniques Used: Activation Assay, Mutagenesis, Purification, Activity Assay, Binding Assay

16) Product Images from "The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells"

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201208150

Morg1 and Cdc42 interact with Par6 in a mutually exclusive manner. (A) Schematic structure of Par6β and its truncated proteins used in the present study. (B, C, E, and H) Proteins in the lysate of COS-7 cells expressing indicated proteins (Cell lysate) were immunoprecipitated (IP) and then analyzed by immunoblot (Blot) with the indicated antibodies. (D) GST–Par6β (126–150, 153–254, or 126–254) or GST alone was incubated with MBP–Morg1 or MBP alone, and pulled down with glutathione-Sepharose 4B beads, followed by SDS-PAGE analysis with Coomassie brilliant blue (CBB) staining or immunoblot with anti-MBP antibodies. (F) GST–Crb3-(84–120) or GST–Crb3-(84–116) was incubated with MBP–Par6β-(126–254), and analyzed as in D. (G) GST–Par6β-(126–254) or GST alone was incubated with MBP–Morg1 in the presence of a two- or sixfold molar excess of Cdc42 (Q61L) or Cdc42 (T17N) relative to Morg1 (Ratio). Proteins pulled down with glutathione-Sepharose 4B beads were subjected to SDS-PAGE and stained with CBB.
Figure Legend Snippet: Morg1 and Cdc42 interact with Par6 in a mutually exclusive manner. (A) Schematic structure of Par6β and its truncated proteins used in the present study. (B, C, E, and H) Proteins in the lysate of COS-7 cells expressing indicated proteins (Cell lysate) were immunoprecipitated (IP) and then analyzed by immunoblot (Blot) with the indicated antibodies. (D) GST–Par6β (126–150, 153–254, or 126–254) or GST alone was incubated with MBP–Morg1 or MBP alone, and pulled down with glutathione-Sepharose 4B beads, followed by SDS-PAGE analysis with Coomassie brilliant blue (CBB) staining or immunoblot with anti-MBP antibodies. (F) GST–Crb3-(84–120) or GST–Crb3-(84–116) was incubated with MBP–Par6β-(126–254), and analyzed as in D. (G) GST–Par6β-(126–254) or GST alone was incubated with MBP–Morg1 in the presence of a two- or sixfold molar excess of Cdc42 (Q61L) or Cdc42 (T17N) relative to Morg1 (Ratio). Proteins pulled down with glutathione-Sepharose 4B beads were subjected to SDS-PAGE and stained with CBB.

Techniques Used: Expressing, Immunoprecipitation, Incubation, SDS Page, Staining

Cdc42 facilitates Par6 binding to Crb3. (A and C) Proteins in the lysate of COS-7 cells expressing indicated proteins (Cell lysate) were immunoprecipitated (IP) and then analyzed by immunoblot (Blot) with the indicated antibodies. In C, the arrow and arrowhead indicate the positions of FLAG–Crb3 and Myc–Par6, respectively; Single and double asterisks denote the heavy and light chains of IgG, respectively. (B) GST–Crb3-(84–120 or 84–116) was incubated with MBP–Par6β-(126–254) in the presence of Cdc42 (Q61L or T17N), and pulled down with glutathione-Sepharose 4B beads, followed by SDS-PAGE analysis with CBB staining.
Figure Legend Snippet: Cdc42 facilitates Par6 binding to Crb3. (A and C) Proteins in the lysate of COS-7 cells expressing indicated proteins (Cell lysate) were immunoprecipitated (IP) and then analyzed by immunoblot (Blot) with the indicated antibodies. In C, the arrow and arrowhead indicate the positions of FLAG–Crb3 and Myc–Par6, respectively; Single and double asterisks denote the heavy and light chains of IgG, respectively. (B) GST–Crb3-(84–120 or 84–116) was incubated with MBP–Par6β-(126–254) in the presence of Cdc42 (Q61L or T17N), and pulled down with glutathione-Sepharose 4B beads, followed by SDS-PAGE analysis with CBB staining.

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

17) Product Images from "The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth"

Article Title: The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth

Journal: Nature Communications

doi: 10.1038/ncomms4351

Isolation of DUSP26 as a FADD phosphatase and an AK2-binding partner. ( a ) HEK293T cells were co-transfected with p-FADD-HA and each phosphatase cDNA for 24 h, cell extracts were subjected to western blotting using anti-p-FADD and anti-FADD antibodies and to phosphatase assays with pNPP. Bars represent mean±s.d. ( n =3). ( b ) HEK293T cells were co-transfected with FADD-HA and pcDNA3, Flag-DUSP26 or Flag-DUSP26 C152S for 24 h, and cell extracts were then analysed by western blotting. ( c ) HeLa cells were transfected with pSuper, pDUSP26 or pDUSP26 shRNA for 36 h, after which cell extracts were subjected to western blotting using anti-p-FADD, anti-FADD and anti-α-tubulin antibodies. Total RNAs were purified and analysed with RT–PCR using DUSP26- or GAPDH-specific synthetic oligonucleotides as primers. ( d ) HEK293T cells were transfected with p3 × Flag or p3 × Flag-DUSP26 for 36 h, and cell extracts were prepared and subjected to the pull-down assay using anti-FLAG agarose-beads. The bound proteins were resolved by SDS–PAGE, stained with Coomassie-blue, and analysed by LC-MS/MS or analysed by western blotting using the indicated antibodies. NS indicates non-specific signal. ( e ) HEK293T cells were co-transfected with pAK2-HA and pFlag-DUSP26 for 36 h and cell extracts were subjected to immunoprecipitation (IP) assay using anti-HA or anti-Flag antibody. Whole-cell lysates and the immunoprecipitates were probed by western blotting with anti-FLAG or anti-HA antibody. ( f ) HeLa cell extracts were subjected to IP analysis using anti-AK2 or anti-DUSP26 antibodies and then the immunoprecipitates were analysed by western blotting with the indicated antibodies. ( g ) AK2 over-expression enhances the binding of DUSP26 to FADD. HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either GFP or AK2-GFP for 36 h, after which cells extracts were subjected to IP analysis using anti-HA antibody. Expression levels of Flag-DUSP26, FADD-HA, GFP and AK2-GFP in whole-cell lysates were examined by western blotting with the indicated antibodies. ( h ) HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either pSuper or AK2 shRNA for 36 h. Cell lysates were subjected to IP analysis with anti-HA antibody, and expression levels of Flag-DUSP26, FADD-HA and AK2 in whole-cell lysates were examined by western blotting.
Figure Legend Snippet: Isolation of DUSP26 as a FADD phosphatase and an AK2-binding partner. ( a ) HEK293T cells were co-transfected with p-FADD-HA and each phosphatase cDNA for 24 h, cell extracts were subjected to western blotting using anti-p-FADD and anti-FADD antibodies and to phosphatase assays with pNPP. Bars represent mean±s.d. ( n =3). ( b ) HEK293T cells were co-transfected with FADD-HA and pcDNA3, Flag-DUSP26 or Flag-DUSP26 C152S for 24 h, and cell extracts were then analysed by western blotting. ( c ) HeLa cells were transfected with pSuper, pDUSP26 or pDUSP26 shRNA for 36 h, after which cell extracts were subjected to western blotting using anti-p-FADD, anti-FADD and anti-α-tubulin antibodies. Total RNAs were purified and analysed with RT–PCR using DUSP26- or GAPDH-specific synthetic oligonucleotides as primers. ( d ) HEK293T cells were transfected with p3 × Flag or p3 × Flag-DUSP26 for 36 h, and cell extracts were prepared and subjected to the pull-down assay using anti-FLAG agarose-beads. The bound proteins were resolved by SDS–PAGE, stained with Coomassie-blue, and analysed by LC-MS/MS or analysed by western blotting using the indicated antibodies. NS indicates non-specific signal. ( e ) HEK293T cells were co-transfected with pAK2-HA and pFlag-DUSP26 for 36 h and cell extracts were subjected to immunoprecipitation (IP) assay using anti-HA or anti-Flag antibody. Whole-cell lysates and the immunoprecipitates were probed by western blotting with anti-FLAG or anti-HA antibody. ( f ) HeLa cell extracts were subjected to IP analysis using anti-AK2 or anti-DUSP26 antibodies and then the immunoprecipitates were analysed by western blotting with the indicated antibodies. ( g ) AK2 over-expression enhances the binding of DUSP26 to FADD. HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either GFP or AK2-GFP for 36 h, after which cells extracts were subjected to IP analysis using anti-HA antibody. Expression levels of Flag-DUSP26, FADD-HA, GFP and AK2-GFP in whole-cell lysates were examined by western blotting with the indicated antibodies. ( h ) HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either pSuper or AK2 shRNA for 36 h. Cell lysates were subjected to IP analysis with anti-HA antibody, and expression levels of Flag-DUSP26, FADD-HA and AK2 in whole-cell lysates were examined by western blotting.

Techniques Used: Isolation, Binding Assay, Transfection, Western Blot, shRNA, Purification, Reverse Transcription Polymerase Chain Reaction, Pull Down Assay, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunoprecipitation, Over Expression, Expressing

18) Product Images from "Autophosphorylation of a Bacterial Serine/Threonine Kinase, AfsK, Is Inhibited by KbpA, an AfsK-Binding Protein"

Article Title: Autophosphorylation of a Bacterial Serine/Threonine Kinase, AfsK, Is Inhibited by KbpA, an AfsK-Binding Protein

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.19.5506-5512.2001

Interaction of KbpA and AfsK. (A) The pull-down of GST-AfsK with glutathione-Sepharose coprecipitated TRX-KbpA, which was detected with S protein by Western blotting (immunoblotting [IB]) (lane 4). TRX-KbpA was not recovered when GST or GST-AfsR was used. The pull-down of GST-AfsR (132 kDa), GST-AfsK (110 kDa), and GST (28 kDa) with glutathione-Sepharose was apparent by Western blotting with the antibody for GST (α-GST). The small protein found in lanes 5 and 6 is a degradation product derived from GST-AfsR. (B) Autophosphorylation of TRX-KΔC wt (51 kDa) and phosphorylation of GST-AfsR by TRX-KΔC wt . TRX-KΔC wt (5 μg) was incubated at 30°C for 10 min in the presence of [γ- 32 P]ATP, subjected to SDS-PAGE, and analyzed by autoradiography. GST-AfsR (3 μg) in the reaction mixture was also phosphorylated. Neither autophosphorylation nor phosphorylation of GST-AfsR occurred for TRX-KΔC K44A . (C) SDS-PAGE of the TRX-KΔC proteins labeled in vivo. Coomassie brilliant blue (CBB) staining revealed smeared bands for TRX-KΔC wt , as indicated by an asterisk, which represent phosphorylated forms of TRX-KΔC wt , as found by autoradiography. (D) The pull-down of GST-KbpA (54 kDa) with glutathione-Sepharose coprecipitated TRX-KΔC wt (lane 3) and TRX-KΔC K44A (lane 4) without recovering smeared, phosphorylated forms of TRX-KΔC wt . The small protein recovered by anti-GST antibody in lanes 3 and 4 is a degradation product. GST itself did not pull down the TRX-KΔC proteins (lanes 1 and 2). TRX-KΔC wt gave smeared bands (lane 5), but TRX-KΔC K44A did not (lane 6).
Figure Legend Snippet: Interaction of KbpA and AfsK. (A) The pull-down of GST-AfsK with glutathione-Sepharose coprecipitated TRX-KbpA, which was detected with S protein by Western blotting (immunoblotting [IB]) (lane 4). TRX-KbpA was not recovered when GST or GST-AfsR was used. The pull-down of GST-AfsR (132 kDa), GST-AfsK (110 kDa), and GST (28 kDa) with glutathione-Sepharose was apparent by Western blotting with the antibody for GST (α-GST). The small protein found in lanes 5 and 6 is a degradation product derived from GST-AfsR. (B) Autophosphorylation of TRX-KΔC wt (51 kDa) and phosphorylation of GST-AfsR by TRX-KΔC wt . TRX-KΔC wt (5 μg) was incubated at 30°C for 10 min in the presence of [γ- 32 P]ATP, subjected to SDS-PAGE, and analyzed by autoradiography. GST-AfsR (3 μg) in the reaction mixture was also phosphorylated. Neither autophosphorylation nor phosphorylation of GST-AfsR occurred for TRX-KΔC K44A . (C) SDS-PAGE of the TRX-KΔC proteins labeled in vivo. Coomassie brilliant blue (CBB) staining revealed smeared bands for TRX-KΔC wt , as indicated by an asterisk, which represent phosphorylated forms of TRX-KΔC wt , as found by autoradiography. (D) The pull-down of GST-KbpA (54 kDa) with glutathione-Sepharose coprecipitated TRX-KΔC wt (lane 3) and TRX-KΔC K44A (lane 4) without recovering smeared, phosphorylated forms of TRX-KΔC wt . The small protein recovered by anti-GST antibody in lanes 3 and 4 is a degradation product. GST itself did not pull down the TRX-KΔC proteins (lanes 1 and 2). TRX-KΔC wt gave smeared bands (lane 5), but TRX-KΔC K44A did not (lane 6).

Techniques Used: Western Blot, Derivative Assay, Incubation, SDS Page, Autoradiography, Labeling, In Vivo, Staining

19) Product Images from "Involvement of Alpha-PAK-Interacting Exchange Factor in the PAK1-c-Jun NH2-Terminal Kinase 1 Activation and Apoptosis Induced by Benzo[a]pyrene"

Article Title: Involvement of Alpha-PAK-Interacting Exchange Factor in the PAK1-c-Jun NH2-Terminal Kinase 1 Activation and Apoptosis Induced by Benzo[a]pyrene

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.20.6796-6807.2001

αPIX and the JNK pathway kinases facilitate the apoptotic cell death induced by B(a)P. (A) αPIX (ΔCH) and PAK1 (T423E) accelerate B(a)P-induced apoptotic cell death. HeLa cells were cotransfected with an eGFP expression vector and test plasmids as indicated. At 24 h posttransfection, the cells were placed in medium with 0.5% FBS and incubated for a further 12 h. They were then treated with 10 μ M B(a)P for 24 h or left untreated and were subjected to analysis. (Top) Quantitation of apoptotic cell death by using nuclear morphology. (Center) DNA integrity of transfected cells. Total DNA from the transfected samples was isolated, and an equal amount of DNA from each was separated on a 1.5% agarose gel. (Bottom) Expression levels of myc-tagged αPIX and PAK1, HA-tagged SEK1, and Flag-tagged Bcl-2 proteins as determined by immunoblot analysis. (B) αPIX (ΔSH3), PAK1 (K299R) and SEK1 (AL) inhibit apoptotic cell death induced by B(a)P. HeLa cells were cotransfected with an eGFP expression vector and test plasmids as indicated. At 36 h posttransfection, the cells were treated with 10μ M B(a)P for 36 h and subjected to analysis. (C) SEK1 (AL) blocks apoptosis accelerated by αPIX (ΔCH) in cells treated with B(a)P. HeLa cells were cotransfected with an eGFP expression vector and plasmids encoding myc-tagged αPIX (ΔCH) and HA-tagged SEK1 (AL) or Flag-tagged Bcl-2 as indicated. The total amount of transfected DNA was made constant by adding vector pCS2+ DNA. At 36 h posttransfection, the cells were treated with 10 μM B(a)P for 24 h and subjected to analysis. (D) The caspase inhibitor Z-Asp-CH2-DCB inhibits apoptosis accelerated by αPIX (Δ CH) in cells treated with B(a)P. HeLa cells were cotransfected with an eGFP expression vector and plasmids encoding myc-tagged αPIX (Δ CH) or mock vector as indicated. At 36 h posttransfection, the cells were preincubated with Z-Asp-CH2-DCB (50 μM) or vehicle (DMSO) for 90 min and treated with 10 μ M B(a)P for 24 h. Then the cells were harvested and subjected to analysis. Similar results were obtained in three independent experiments. The data shown are the mean and standard deviation for three independent experiments. SEK1 (AL), SEK1 (K220A, K224L).
Figure Legend Snippet: αPIX and the JNK pathway kinases facilitate the apoptotic cell death induced by B(a)P. (A) αPIX (ΔCH) and PAK1 (T423E) accelerate B(a)P-induced apoptotic cell death. HeLa cells were cotransfected with an eGFP expression vector and test plasmids as indicated. At 24 h posttransfection, the cells were placed in medium with 0.5% FBS and incubated for a further 12 h. They were then treated with 10 μ M B(a)P for 24 h or left untreated and were subjected to analysis. (Top) Quantitation of apoptotic cell death by using nuclear morphology. (Center) DNA integrity of transfected cells. Total DNA from the transfected samples was isolated, and an equal amount of DNA from each was separated on a 1.5% agarose gel. (Bottom) Expression levels of myc-tagged αPIX and PAK1, HA-tagged SEK1, and Flag-tagged Bcl-2 proteins as determined by immunoblot analysis. (B) αPIX (ΔSH3), PAK1 (K299R) and SEK1 (AL) inhibit apoptotic cell death induced by B(a)P. HeLa cells were cotransfected with an eGFP expression vector and test plasmids as indicated. At 36 h posttransfection, the cells were treated with 10μ M B(a)P for 36 h and subjected to analysis. (C) SEK1 (AL) blocks apoptosis accelerated by αPIX (ΔCH) in cells treated with B(a)P. HeLa cells were cotransfected with an eGFP expression vector and plasmids encoding myc-tagged αPIX (ΔCH) and HA-tagged SEK1 (AL) or Flag-tagged Bcl-2 as indicated. The total amount of transfected DNA was made constant by adding vector pCS2+ DNA. At 36 h posttransfection, the cells were treated with 10 μM B(a)P for 24 h and subjected to analysis. (D) The caspase inhibitor Z-Asp-CH2-DCB inhibits apoptosis accelerated by αPIX (Δ CH) in cells treated with B(a)P. HeLa cells were cotransfected with an eGFP expression vector and plasmids encoding myc-tagged αPIX (Δ CH) or mock vector as indicated. At 36 h posttransfection, the cells were preincubated with Z-Asp-CH2-DCB (50 μM) or vehicle (DMSO) for 90 min and treated with 10 μ M B(a)P for 24 h. Then the cells were harvested and subjected to analysis. Similar results were obtained in three independent experiments. The data shown are the mean and standard deviation for three independent experiments. SEK1 (AL), SEK1 (K220A, K224L).

Techniques Used: Expressing, Plasmid Preparation, Incubation, Quantitation Assay, Transfection, Isolation, Agarose Gel Electrophoresis, Standard Deviation

20) Product Images from "IKKα controls ATG16L1 degradation to prevent ER stress during inflammation"

Article Title: IKKα controls ATG16L1 degradation to prevent ER stress during inflammation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20161867

IKKα phosphorylates ATG16L1. (A) Coimmunoprecipitation (Co-IP) and immunoblot (IB) analysis showing interaction of FLAG-IKKα and mCherry-ATG16L1. HEK293T cells were transfected as indicated, and lysates were subjected to coimmunoprecipitation assays. Immunoblots from 40-µg input were used to examine protein expression levels. (B) Coimmunoprecipitation of endogenous IKKα and ATG16L1 in WT colonic organoids. 600 µg of protein lysates from colon organoids were immunoprecipitated with ATG16L1 or control IgG. The immunoprecipitates and 40 µg of input lysates were analyzed by immunoblot analysis using the indicated antibodies. Data are representative of two experiments. (C) HEK293T cells were transfected with FLAG-IKKα, and whole-cell extracts were subjected to a pull down assay using various forms of GST-ATG16L1 and GST-Sepharose beads. The bead-bound proteins were analyzed by immunoblot analysis using anti-FLAG antibody. Data are representative of two experiments. (D) Schematic representation of the full-length ATG16L1 and different construct domains used for the expression of GST-fusion proteins. (E) HEK293T cells were transfected with FLAG-IKKα Wt or FLAG-IKKα AA . After 48 h, the cells were collected and lysed, and whole-cell extracts were subjected to immunoprecipitation using protein A/G plus agarose beads and anti-FLAG antibody. Then, immunoprecipitates were subjected to in vitro kinase assay with GST-ATG16L1(231–352). The FLAG immunoblot shows equal amounts of immunopurified FLAG-IKKα Wt or FLAG-IKKα AA . Phosphorylation of GST-ATG16L1(231–352) was confirmed by mass spectrometry. Data are representative of three experiments. (F) Mass spectrometric fragment ion scan of the peptide corresponding to phosphorylated serine 278 in ATG16L1. Data are representative of two experiments. (G) In vitro kinase assay of FLAG-IKKα Wt overexpressing HEK293T cells and different point-mutated GST-ATG16L1(231–352) substrates. Phosphorylated GST-ATG16L1(231–352) was visualized by autoradiography. Ponceau S staining shows the equal amounts of GST-ATG16L1 substrates. Data are representative of two experiments. EV, empty vector. (H) Endogenous IKK complex was immunoprecipitated using anti-IKKγ from untreated or 20 ng/ml TNF–treated (15 min) colonic organoids isolated from Ikkα Wt/Wt and Ikkα AA/AA mice and then subjected to in vitro kinase assay using GST-ATG16L1(231–352) as a substrate. Phosphorylated GST-ATG16L1(231–352) was visualized by autoradiography. Ponceau S staining and immunoblotting against IKKα and IKKβ show the equal amount of GST-ATG16L1 and immunoprecipitation efficiency, respectively. Data are representative of two experiments. (I) ATG16L1 proteolytic cleavage was assessed by immunoblot analysis in HeLa cells expressing mCherry-ATG16L1 Wt and mutants. HeLa cells were pretreated for 1 h with DMSO or 10 µM pan-caspase inhibitor (zVADfmk), followed by TNF stimulation (20 ng/ml) in the presence of 10 µg/ml cycloheximide (CHX) for 3 h. (J) Caspase 3–mediated in vitro ATG16L1 cleavage was assessed by immunoblot analysis. mCherry-ATG16L1 Wt and mutants were immunoprecipitated from HEK293T cells. Then, immunoprecipitates were subjected to in vitro cleavage assay using recombinant active caspase 3. Data are representative of two experiments. (I and J) CL, cleaved; FL, full length. (K) ATG16L1 proteolytic cleavage was assessed by immunoblot analysis in colonic organoids. Organoids were pretreated with DMSO or pan-caspase inhibitor (zVADfmk), followed by stimulation with NOD ligands, 20 µg/ml L-18MDP, and 20 µg/ml C12-iE-DAP, in the presence of 10 µg/ml cycloheximide for 3.5 h. Data are representative of two experiments. cl., cleaved. (L) Endogenous IKK complex was immunoprecipitated from untreated or L18-MDP–treated (20 µg/ml for 15 min) colonic organoids and then subjected to in vitro kinase assay as described in H. Data are representative of two experiments. (G, H, and L) Single asterisks indicate nonspecific signal determined by mass spectrometry.
Figure Legend Snippet: IKKα phosphorylates ATG16L1. (A) Coimmunoprecipitation (Co-IP) and immunoblot (IB) analysis showing interaction of FLAG-IKKα and mCherry-ATG16L1. HEK293T cells were transfected as indicated, and lysates were subjected to coimmunoprecipitation assays. Immunoblots from 40-µg input were used to examine protein expression levels. (B) Coimmunoprecipitation of endogenous IKKα and ATG16L1 in WT colonic organoids. 600 µg of protein lysates from colon organoids were immunoprecipitated with ATG16L1 or control IgG. The immunoprecipitates and 40 µg of input lysates were analyzed by immunoblot analysis using the indicated antibodies. Data are representative of two experiments. (C) HEK293T cells were transfected with FLAG-IKKα, and whole-cell extracts were subjected to a pull down assay using various forms of GST-ATG16L1 and GST-Sepharose beads. The bead-bound proteins were analyzed by immunoblot analysis using anti-FLAG antibody. Data are representative of two experiments. (D) Schematic representation of the full-length ATG16L1 and different construct domains used for the expression of GST-fusion proteins. (E) HEK293T cells were transfected with FLAG-IKKα Wt or FLAG-IKKα AA . After 48 h, the cells were collected and lysed, and whole-cell extracts were subjected to immunoprecipitation using protein A/G plus agarose beads and anti-FLAG antibody. Then, immunoprecipitates were subjected to in vitro kinase assay with GST-ATG16L1(231–352). The FLAG immunoblot shows equal amounts of immunopurified FLAG-IKKα Wt or FLAG-IKKα AA . Phosphorylation of GST-ATG16L1(231–352) was confirmed by mass spectrometry. Data are representative of three experiments. (F) Mass spectrometric fragment ion scan of the peptide corresponding to phosphorylated serine 278 in ATG16L1. Data are representative of two experiments. (G) In vitro kinase assay of FLAG-IKKα Wt overexpressing HEK293T cells and different point-mutated GST-ATG16L1(231–352) substrates. Phosphorylated GST-ATG16L1(231–352) was visualized by autoradiography. Ponceau S staining shows the equal amounts of GST-ATG16L1 substrates. Data are representative of two experiments. EV, empty vector. (H) Endogenous IKK complex was immunoprecipitated using anti-IKKγ from untreated or 20 ng/ml TNF–treated (15 min) colonic organoids isolated from Ikkα Wt/Wt and Ikkα AA/AA mice and then subjected to in vitro kinase assay using GST-ATG16L1(231–352) as a substrate. Phosphorylated GST-ATG16L1(231–352) was visualized by autoradiography. Ponceau S staining and immunoblotting against IKKα and IKKβ show the equal amount of GST-ATG16L1 and immunoprecipitation efficiency, respectively. Data are representative of two experiments. (I) ATG16L1 proteolytic cleavage was assessed by immunoblot analysis in HeLa cells expressing mCherry-ATG16L1 Wt and mutants. HeLa cells were pretreated for 1 h with DMSO or 10 µM pan-caspase inhibitor (zVADfmk), followed by TNF stimulation (20 ng/ml) in the presence of 10 µg/ml cycloheximide (CHX) for 3 h. (J) Caspase 3–mediated in vitro ATG16L1 cleavage was assessed by immunoblot analysis. mCherry-ATG16L1 Wt and mutants were immunoprecipitated from HEK293T cells. Then, immunoprecipitates were subjected to in vitro cleavage assay using recombinant active caspase 3. Data are representative of two experiments. (I and J) CL, cleaved; FL, full length. (K) ATG16L1 proteolytic cleavage was assessed by immunoblot analysis in colonic organoids. Organoids were pretreated with DMSO or pan-caspase inhibitor (zVADfmk), followed by stimulation with NOD ligands, 20 µg/ml L-18MDP, and 20 µg/ml C12-iE-DAP, in the presence of 10 µg/ml cycloheximide for 3.5 h. Data are representative of two experiments. cl., cleaved. (L) Endogenous IKK complex was immunoprecipitated from untreated or L18-MDP–treated (20 µg/ml for 15 min) colonic organoids and then subjected to in vitro kinase assay as described in H. Data are representative of two experiments. (G, H, and L) Single asterisks indicate nonspecific signal determined by mass spectrometry.

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Western Blot, Expressing, Immunoprecipitation, Pull Down Assay, Construct, In Vitro, Kinase Assay, Mass Spectrometry, Autoradiography, Staining, Plasmid Preparation, Isolation, Mouse Assay, Cleavage Assay, Recombinant

21) Product Images from "Functional domains of yeast hexokinase 2"

Article Title: Functional domains of yeast hexokinase 2

Journal: Biochemical Journal

doi: 10.1042/BJ20100663

Interaction of Hxk2 wca and Hxk2 wrf with Mig1 ( A ) In vivo co-immunoprecipitation of HA–Mig1 with Hxk2 wca and Hxk2 wrf . The W303.1A, W303.1A wca and W303.1A wrf strains, transformed with plasmid pWS93/Mig1 which encoded an HA-tagged Mig1 protein, were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody (lanes 1–6) or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Mig1 was visualized on a Western blot with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody. ( B ) Mig1 phosphorylation in response to Hxk2 wca and Hxk2 wrf availability. Cells, from wild-type (WT), Hxk2 wca and Hxk2 wrf strains transformed with the HA–Mig1 construct (plasmid pWS93/Mig1), were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The Mig1 protein was detected from total cell extracts by SDS/12% PAGE followed by immunoblotting with an anti-HA antibody. The phosphorylated forms of Mig1 are indicated as HA-Mig1-P (phosphorylated) and HA-Mig1 (dephosphorylated). ( C ) GST pull-down assays of the interaction of Hxk2 wca and Hxk2 wrf proteins with Mig1. A GST–Mig1 fusion protein was purified on glutathione–Sepharose columns. Equal amounts of GST–Mig1 were incubated with cell extracts from a wild-type strain (W303.1A) and the mutant strains W303.1A wca and W303.1A wrf . The yeast strains were grown in YEPG medium until a D 600 of 0.6 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. For the control samples, GST protein was also incubated with the H-Glc and L-Glc cell extracts, but no signals were detected (lanes 5 and 6). ( D ) The level of Hxk2 present in the different extracts used in ( C ) was determined by Western blotting using an anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.
Figure Legend Snippet: Interaction of Hxk2 wca and Hxk2 wrf with Mig1 ( A ) In vivo co-immunoprecipitation of HA–Mig1 with Hxk2 wca and Hxk2 wrf . The W303.1A, W303.1A wca and W303.1A wrf strains, transformed with plasmid pWS93/Mig1 which encoded an HA-tagged Mig1 protein, were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody (lanes 1–6) or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Mig1 was visualized on a Western blot with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody. ( B ) Mig1 phosphorylation in response to Hxk2 wca and Hxk2 wrf availability. Cells, from wild-type (WT), Hxk2 wca and Hxk2 wrf strains transformed with the HA–Mig1 construct (plasmid pWS93/Mig1), were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The Mig1 protein was detected from total cell extracts by SDS/12% PAGE followed by immunoblotting with an anti-HA antibody. The phosphorylated forms of Mig1 are indicated as HA-Mig1-P (phosphorylated) and HA-Mig1 (dephosphorylated). ( C ) GST pull-down assays of the interaction of Hxk2 wca and Hxk2 wrf proteins with Mig1. A GST–Mig1 fusion protein was purified on glutathione–Sepharose columns. Equal amounts of GST–Mig1 were incubated with cell extracts from a wild-type strain (W303.1A) and the mutant strains W303.1A wca and W303.1A wrf . The yeast strains were grown in YEPG medium until a D 600 of 0.6 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. For the control samples, GST protein was also incubated with the H-Glc and L-Glc cell extracts, but no signals were detected (lanes 5 and 6). ( D ) The level of Hxk2 present in the different extracts used in ( C ) was determined by Western blotting using an anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.

Techniques Used: In Vivo, Immunoprecipitation, Transformation Assay, Plasmid Preparation, Selection, Gas Chromatography, Polyacrylamide Gel Electrophoresis, Western Blot, Construct, Purification, Incubation, Mutagenesis

22) Product Images from "Recruitment of Cytosolic J-Proteins by TOM Receptors Promotes Mitochondrial Protein Biogenesis"

Article Title: Recruitment of Cytosolic J-Proteins by TOM Receptors Promotes Mitochondrial Protein Biogenesis

Journal: Cell Reports

doi: 10.1016/j.celrep.2018.10.083

Xdj1 Delivers Precursor Proteins to the Tom22 Receptor (A and B) 35 S-labeled precursors were incubated with glutathione Sepharose coupled with GST, GST Xdj1, GST Ydj1, or GST Djp1. Load and elution fractions were analyzed by SDS-PAGE and autoradiography. (A) Load 0.7%; elution was 33%. (B) Input, translation product (TLP); elution was 50%. (C) [ 35 S]b 2 -DHFR and [ 35 S]Su9-DHFR precursors were incubated with GST-tagged J-proteins, followed by import into isolated wild-type mitochondria. The import reaction was analyzed by SDS-PAGE and autoradiography. p, precursor; i, intermediate; m, mature. Quantification of mature-sized proteins is shown, mean values ± SEM (n = 3–4); the import after 12 min in the presence of GST was set to 100% (control). (D) Left panel, [ 35 S]Oxa1 precursor was incubated with glutathione Sepharose coupled with GST Xdj1. Bound proteins were eluted with increasing amounts of His-tagged cytosolic domain (CD) of Tom22. Load was 1%; elution was 25%. Second panel, [ 35 S]Oxa1 was incubated with glutathione Sepharose coupled with GST Xdj1. Bound proteins were incubated in the presence or absence of His-tagged Tom22 CD . The eluted proteins were purified via Ni-NTA and analyzed by SDS-PAGE and autoradiography. Third panel, [ 35 S]Oxa1 was incubated in the presence or absence of GST Xdj1, and the binding to His-tagged Tom22 CD was analyzed by SDS-PAGE and autoradiography. Input was 1%; elution was 100%. Right panel, [ 35 S]Oxa1 was incubated with glutathione Sepharose coupled with GST Xdj1. Bound proteins were eluted, imported into isolated mitochondria, and analyzed by SDS-PAGE and autoradiography. p, precursor; m, mature.
Figure Legend Snippet: Xdj1 Delivers Precursor Proteins to the Tom22 Receptor (A and B) 35 S-labeled precursors were incubated with glutathione Sepharose coupled with GST, GST Xdj1, GST Ydj1, or GST Djp1. Load and elution fractions were analyzed by SDS-PAGE and autoradiography. (A) Load 0.7%; elution was 33%. (B) Input, translation product (TLP); elution was 50%. (C) [ 35 S]b 2 -DHFR and [ 35 S]Su9-DHFR precursors were incubated with GST-tagged J-proteins, followed by import into isolated wild-type mitochondria. The import reaction was analyzed by SDS-PAGE and autoradiography. p, precursor; i, intermediate; m, mature. Quantification of mature-sized proteins is shown, mean values ± SEM (n = 3–4); the import after 12 min in the presence of GST was set to 100% (control). (D) Left panel, [ 35 S]Oxa1 precursor was incubated with glutathione Sepharose coupled with GST Xdj1. Bound proteins were eluted with increasing amounts of His-tagged cytosolic domain (CD) of Tom22. Load was 1%; elution was 25%. Second panel, [ 35 S]Oxa1 was incubated with glutathione Sepharose coupled with GST Xdj1. Bound proteins were incubated in the presence or absence of His-tagged Tom22 CD . The eluted proteins were purified via Ni-NTA and analyzed by SDS-PAGE and autoradiography. Third panel, [ 35 S]Oxa1 was incubated in the presence or absence of GST Xdj1, and the binding to His-tagged Tom22 CD was analyzed by SDS-PAGE and autoradiography. Input was 1%; elution was 100%. Right panel, [ 35 S]Oxa1 was incubated with glutathione Sepharose coupled with GST Xdj1. Bound proteins were eluted, imported into isolated mitochondria, and analyzed by SDS-PAGE and autoradiography. p, precursor; m, mature.

Techniques Used: Labeling, Incubation, SDS Page, Autoradiography, Isolation, Purification, Binding Assay

Xdj1 Binds to the TOM Complex via Tom22, whereas Djp1 Binds to Tom70 (A) Tom22 His and wild-type (WT) mitochondria were subjected to affinity purification via Ni-NTA agarose. Potential interaction partners of Tom22 were identified by SILAC-based quantitative mass spectrometry. Depicted are the mean log 10 Tom22 His / WT ratios and the corresponding p values (–log 10 -transformed; n ≥ 2). See Table S1 for a complete list of interactors. (B) Yeast cells expressing Xdj1 GFP were stained with MitoTracker Deep Red and analyzed by fluorescence microscopy. Z-slices of the green fluorescence of GFP, the red fluorescence of MitoTracker, and merged images are shown. Scale bar, 5 μm. (C) 35 S-labeled Xdj1 was incubated with Ni-NTA agarose and with Ni-NTA coated with the His-tagged cytosolic domains (CD) of Tom20, Tom22, or Tom70. Load (2.5%) and elution (100%) were analyzed by SDS-PAGE and autoradiography. Asterisk, non-specific band. (D) Tom22 CD was incubated with glutathione columns coated with GST, GST Xdj1, GST Ydj1, or GST Djp1. Input (2%) and elution (50%) were analyzed by SDS-PAGE and Coomassie blue staining. (E) Xdj1 His was incubated with lysed mitochondria and purified via Ni-NTA agarose. Load (2%) and elution (100%) were analyzed by blue native electrophoresis and immunodetection. (F) [ 35 S]Xdj1 was incubated with the indicated mitochondria, followed by anti-HA chromatography. Load (2%) and elution (100%) were analyzed by SDS-PAGE, immunodetection, and autography. (G) [ 35 S]Xdj1 was incubated with tom22 Δ, tom20 Δ, or tom70 Δ mitochondria and their corresponding WT mitochondria. (H) Lysed mitochondria were incubated with glutathione Sepharose coated with GST, GST Xdj1, GST Ydj1, or GST Djp1. Load (1%) and elution (100%) were analyzed by SDS-PAGE and immunodetection. (I) Tom70 CD was incubated with glutathione Sepharose coupled with GST or GST Djp1. Load (5%) and elution (50%) were analyzed by SDS-PAGE and Coomassie blue staining. See also Figures S1 and S2 and Table S1 .
Figure Legend Snippet: Xdj1 Binds to the TOM Complex via Tom22, whereas Djp1 Binds to Tom70 (A) Tom22 His and wild-type (WT) mitochondria were subjected to affinity purification via Ni-NTA agarose. Potential interaction partners of Tom22 were identified by SILAC-based quantitative mass spectrometry. Depicted are the mean log 10 Tom22 His / WT ratios and the corresponding p values (–log 10 -transformed; n ≥ 2). See Table S1 for a complete list of interactors. (B) Yeast cells expressing Xdj1 GFP were stained with MitoTracker Deep Red and analyzed by fluorescence microscopy. Z-slices of the green fluorescence of GFP, the red fluorescence of MitoTracker, and merged images are shown. Scale bar, 5 μm. (C) 35 S-labeled Xdj1 was incubated with Ni-NTA agarose and with Ni-NTA coated with the His-tagged cytosolic domains (CD) of Tom20, Tom22, or Tom70. Load (2.5%) and elution (100%) were analyzed by SDS-PAGE and autoradiography. Asterisk, non-specific band. (D) Tom22 CD was incubated with glutathione columns coated with GST, GST Xdj1, GST Ydj1, or GST Djp1. Input (2%) and elution (50%) were analyzed by SDS-PAGE and Coomassie blue staining. (E) Xdj1 His was incubated with lysed mitochondria and purified via Ni-NTA agarose. Load (2%) and elution (100%) were analyzed by blue native electrophoresis and immunodetection. (F) [ 35 S]Xdj1 was incubated with the indicated mitochondria, followed by anti-HA chromatography. Load (2%) and elution (100%) were analyzed by SDS-PAGE, immunodetection, and autography. (G) [ 35 S]Xdj1 was incubated with tom22 Δ, tom20 Δ, or tom70 Δ mitochondria and their corresponding WT mitochondria. (H) Lysed mitochondria were incubated with glutathione Sepharose coated with GST, GST Xdj1, GST Ydj1, or GST Djp1. Load (1%) and elution (100%) were analyzed by SDS-PAGE and immunodetection. (I) Tom70 CD was incubated with glutathione Sepharose coupled with GST or GST Djp1. Load (5%) and elution (50%) were analyzed by SDS-PAGE and Coomassie blue staining. See also Figures S1 and S2 and Table S1 .

Techniques Used: Affinity Purification, Mass Spectrometry, Transformation Assay, Expressing, Staining, Fluorescence, Microscopy, Labeling, Incubation, SDS Page, Autoradiography, Purification, Electrophoresis, Immunodetection, Chromatography

23) Product Images from "Association of diacylglycerol kinase ? with protein kinase C ?"

Article Title: Association of diacylglycerol kinase ? with protein kinase C ?

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200208120

DGK ζ and PKC α associate with a signaling complex. (A) Lysates from HEK293 cells transiently transfected with PKCα and vector, FLAG-tagged DGKζ (WT or ΔATP), were immunoprecipitated using anti-FLAG or a control antibody (mouse IgG), and then the immunoprecipitates were subjected to immunoblot analysis with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the cell lysates is also shown. (B) Purified PKCα was incubated with purified DGKζ–FLAG bound to anti–FLAG-M2 agarose affinity gel or with affinity gel alone. The beads were washed, and proteins bound to the beads were immunoblotted with anti-PKCα. Input represents 15% of the initial recombinant PKCα used in this experiment. (C) Rat brain extracts were immunoprecipitated with anti-DGKζ or a control antibody (rabbit IgG), followed by immunoblotting with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the rat brain extracts is also shown. (D) Endogenous DGKζ in A172 cell lysates was immunoprecipitated using anti-DGKζ. Normal rabbit IgG was used as a control. The precipitates were subjected to immunoblot analysis with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the A172 cell lysate is shown in the bottom panel.
Figure Legend Snippet: DGK ζ and PKC α associate with a signaling complex. (A) Lysates from HEK293 cells transiently transfected with PKCα and vector, FLAG-tagged DGKζ (WT or ΔATP), were immunoprecipitated using anti-FLAG or a control antibody (mouse IgG), and then the immunoprecipitates were subjected to immunoblot analysis with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the cell lysates is also shown. (B) Purified PKCα was incubated with purified DGKζ–FLAG bound to anti–FLAG-M2 agarose affinity gel or with affinity gel alone. The beads were washed, and proteins bound to the beads were immunoblotted with anti-PKCα. Input represents 15% of the initial recombinant PKCα used in this experiment. (C) Rat brain extracts were immunoprecipitated with anti-DGKζ or a control antibody (rabbit IgG), followed by immunoblotting with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the rat brain extracts is also shown. (D) Endogenous DGKζ in A172 cell lysates was immunoprecipitated using anti-DGKζ. Normal rabbit IgG was used as a control. The precipitates were subjected to immunoblot analysis with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the A172 cell lysate is shown in the bottom panel.

Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Purification, Incubation, Recombinant

A portion of the catalytic domain of DGK ζ is sufficient to bind PKC α . (A) PKCα was transfected into HEK293 cells along with wild-type (WT) DGKζ or deletion mutants of DGKζ (B, H, X, L, ΔM, and Bsu) containing FLAG epitope tags at their COOH termini. DGKζ proteins in the cell lysates were immunoprecipitated with anti-FLAG or a control antibody (mouse IgG), and coimmunoprecipitation of PKCα was detected by immunoblotting. The blot was then stripped and reprobed with anti-DGKζ. Because the DGKζ antibody we used was the NH 2 -terminal anti-peptide rabbit antibody, we could not detect the NH 2 terminus deletion DGKζ mutant L (lane 6). However, we detected DGKζ L protein in the same blot using anti-FLAG antibody (not depicted). Expression of PKCα and DGKζ in the cell lysates is also shown. (B) Purified recombinant PKCα was incubated with the glutathione-sepharose–bound GST (lane 1) or GST fusion proteins that contain either full-length DGKζ (GST–DGKζ, lane 2) or a portion of the catalytic domain of DGKζ (GST–BD, lane 3). The beads were collected by centrifugation, and then the proteins bound to beads were subjected to immunoblot analysis with anti-PKCα. Input represents 5% of initial recombinant PKCα used in this experiment.
Figure Legend Snippet: A portion of the catalytic domain of DGK ζ is sufficient to bind PKC α . (A) PKCα was transfected into HEK293 cells along with wild-type (WT) DGKζ or deletion mutants of DGKζ (B, H, X, L, ΔM, and Bsu) containing FLAG epitope tags at their COOH termini. DGKζ proteins in the cell lysates were immunoprecipitated with anti-FLAG or a control antibody (mouse IgG), and coimmunoprecipitation of PKCα was detected by immunoblotting. The blot was then stripped and reprobed with anti-DGKζ. Because the DGKζ antibody we used was the NH 2 -terminal anti-peptide rabbit antibody, we could not detect the NH 2 terminus deletion DGKζ mutant L (lane 6). However, we detected DGKζ L protein in the same blot using anti-FLAG antibody (not depicted). Expression of PKCα and DGKζ in the cell lysates is also shown. (B) Purified recombinant PKCα was incubated with the glutathione-sepharose–bound GST (lane 1) or GST fusion proteins that contain either full-length DGKζ (GST–DGKζ, lane 2) or a portion of the catalytic domain of DGKζ (GST–BD, lane 3). The beads were collected by centrifugation, and then the proteins bound to beads were subjected to immunoblot analysis with anti-PKCα. Input represents 5% of initial recombinant PKCα used in this experiment.

Techniques Used: Transfection, FLAG-tag, Immunoprecipitation, Mutagenesis, Expressing, Purification, Recombinant, Incubation, Centrifugation

Activation of PKC α impairs its association with DGK ζ . (A) HEK293 cells transfected with PKCα and DGKζ–FLAG were stimulated with PMA or vehicle for 30 min. DGKζ in the cell lysates was immunoprecipitated by anti-FLAG, and coimmunoprecipitation of PKCα was detected by immunoblotting. To inhibit PKC activity, cells were treated with Gö 6983 for 10 min before PMA stimulation. The blot was then stripped and reprobed to detect DGKζ. Expression of DGKζ and PKCα in the cell lysates is also shown. (B) Purified recombinant PKCα was incubated with purified DGKζ–FLAG bound to anti–FLAG-M2 agarose affinity gel or with affinity gel alone in PKC assay buffer (containing phosphatase inhibitors) in the presence or absence of PMA. After 2 h, the beads were washed, and proteins bound to beads were immunoblotted to detect PKCα. Input represents 5% of the initial recombinant PKCα. (C) A172 cells, treated with either 50 ng/ml of PDGF or vehicle for 30 min, were lysed, and then endogenous PKCα proteins were immunoprecipitated with anti-PKCα or normal rabbit IgG as a control. The precipitates were then used for DGK activity assays. To inhibit PKC activity, the cells were treated with Gö 6983 before PDGF stimulation. Data are expressed as the mean ± SEM of three independent experiments. An asterisk indicates P
Figure Legend Snippet: Activation of PKC α impairs its association with DGK ζ . (A) HEK293 cells transfected with PKCα and DGKζ–FLAG were stimulated with PMA or vehicle for 30 min. DGKζ in the cell lysates was immunoprecipitated by anti-FLAG, and coimmunoprecipitation of PKCα was detected by immunoblotting. To inhibit PKC activity, cells were treated with Gö 6983 for 10 min before PMA stimulation. The blot was then stripped and reprobed to detect DGKζ. Expression of DGKζ and PKCα in the cell lysates is also shown. (B) Purified recombinant PKCα was incubated with purified DGKζ–FLAG bound to anti–FLAG-M2 agarose affinity gel or with affinity gel alone in PKC assay buffer (containing phosphatase inhibitors) in the presence or absence of PMA. After 2 h, the beads were washed, and proteins bound to beads were immunoblotted to detect PKCα. Input represents 5% of the initial recombinant PKCα. (C) A172 cells, treated with either 50 ng/ml of PDGF or vehicle for 30 min, were lysed, and then endogenous PKCα proteins were immunoprecipitated with anti-PKCα or normal rabbit IgG as a control. The precipitates were then used for DGK activity assays. To inhibit PKC activity, the cells were treated with Gö 6983 before PDGF stimulation. Data are expressed as the mean ± SEM of three independent experiments. An asterisk indicates P

Techniques Used: Activation Assay, Transfection, Immunoprecipitation, Activity Assay, Expressing, Purification, Recombinant, Incubation

24) Product Images from "A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression"

Article Title: A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression

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

doi: 10.1073/pnas.0400265101

Induction of HIF-1α transactivation by DHR by inhibition of prolyl hydroxylase activity. ( A ) Cells were cotransfected with a luciferase reporter pG5-E1B-LUC and various fusion gene constructs in which the Gal4 DNA binding domain was fused to either HIF-1α TADN or TADC ( Left ). Mutations in constructs are shown in italics. After 24 h, transfected cells were treated with either 6 μM DHR or 6 μM RH and then cultured for 24 h under normoxia (16% O 2 ) or hypoxia (8% O 2 ). Values are given as mean ± SEM of four independent experiments. Student's t test for paired values was performed to determine significant difference. * , 16% O 2 (Control) vs. 8% O 2 . ** , Control vs. DHR; P ≤ 0.05. ( B ) VHL pull-down assay. The GST-TADN-HIF-1α protein was incubated either without extract (-E) or with extract supplemented with cofactors (+E) (see Materials and Methods ) in the presence of either DHR or RH. Glutathione-Sepharose beads and [ 35 S]VHL were added, and bound VHL was recovered, subjected to SDS/PAGE, and visualized. The input remains from directly loaded [ 35 S]VHL. The two bands represent the 213- and 160-aa VHL products. ( C ) Dose-dependent inhibition of HIF prolyl hydroxylase activity by DHR. The GST-TADN-HIF-1α protein or the GST protein were incubated with HepG2 cell extract, cofactors, and 5-[ 14 C]2-oxoglutarate in the presence of various DHR or RH concentrations. Radioactivity associated to succinate was determined. In each experiment, the basal HIF-TADN-dependent activity, which was set at 100%, was determined by subtracting the GST-associated activity. Values are given as mean ± SEM from three independent culture experiments, with each point measured in duplicate.
Figure Legend Snippet: Induction of HIF-1α transactivation by DHR by inhibition of prolyl hydroxylase activity. ( A ) Cells were cotransfected with a luciferase reporter pG5-E1B-LUC and various fusion gene constructs in which the Gal4 DNA binding domain was fused to either HIF-1α TADN or TADC ( Left ). Mutations in constructs are shown in italics. After 24 h, transfected cells were treated with either 6 μM DHR or 6 μM RH and then cultured for 24 h under normoxia (16% O 2 ) or hypoxia (8% O 2 ). Values are given as mean ± SEM of four independent experiments. Student's t test for paired values was performed to determine significant difference. * , 16% O 2 (Control) vs. 8% O 2 . ** , Control vs. DHR; P ≤ 0.05. ( B ) VHL pull-down assay. The GST-TADN-HIF-1α protein was incubated either without extract (-E) or with extract supplemented with cofactors (+E) (see Materials and Methods ) in the presence of either DHR or RH. Glutathione-Sepharose beads and [ 35 S]VHL were added, and bound VHL was recovered, subjected to SDS/PAGE, and visualized. The input remains from directly loaded [ 35 S]VHL. The two bands represent the 213- and 160-aa VHL products. ( C ) Dose-dependent inhibition of HIF prolyl hydroxylase activity by DHR. The GST-TADN-HIF-1α protein or the GST protein were incubated with HepG2 cell extract, cofactors, and 5-[ 14 C]2-oxoglutarate in the presence of various DHR or RH concentrations. Radioactivity associated to succinate was determined. In each experiment, the basal HIF-TADN-dependent activity, which was set at 100%, was determined by subtracting the GST-associated activity. Values are given as mean ± SEM from three independent culture experiments, with each point measured in duplicate.

Techniques Used: Inhibition, Activity Assay, Luciferase, Construct, Binding Assay, Transfection, Cell Culture, Pull Down Assay, Incubation, SDS Page, Radioactivity

25) Product Images from "Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle"

Article Title: Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle

Journal: Nature Communications

doi: 10.1038/ncomms8290

Physical interaction between SPD-1 and CYK-4 sensitive to SPD-1 R83W mutation. ( a ) In vitro translated full-length CYK-4 was pulled down by full-length SPD-1 immobilized on chitin beads via a chitin-binding domain (CBD) tag. ( b ) In vitro translated full-length SPD-1 was pulled down by full-length CYK-4 or the centralspindlin holocomplex (CYK-4/ZEN-4) immobilized on glutathione-Sepharose beads via a glutathione- S -transferase (GST) tag. ( c ) Schematic drawings of SPD-1 and CYK-4. R83W indicates the mutation found in the spd-1(oj5) mutant exhibiting central spindle defects. ( d ) Yeast 2-hybrid assay of the indicated combinations of bait and prey. Growth on histidine-deficient medium containing 3-amino-1,2,4-triazole (–His+3AT) indicates a positive interaction between the bait and prey. ( e ) SPD-1 1-228 fragment with or without the R83W mutation (WT: wild type) was pulled down by CYK-4 constructs expressed as fusion proteins with maltose-binding protein (MBP) and detected with an anti-SPD-1 antibody. The CYK-4 tail region is necessary and, if dimerized, sufficient for efficient binding. ( f ) The R83W mutation does not affect the mobility of the SPD-1 full-length protein in Superdex 200 size-exclusion chromatography (blue: wild type; red: R83W). The elution profile of a mixture of standard proteins (Thy, thyroglobulin; IgG, gamma globulin; Ova, ovalbumin; MyG, myoglobin; VB12, vitamin B12) is shown in grey. ( g , h ) The R83W mutation does not affect the interaction of SPD-1 with microtubules. ( g ) Wild-type and R83W SPD-1 were incubated with microtubules or control buffer and sedimented by ultracentrifugation. P, pellet; S, supernatant; T, total. Increased recovery in the pellet in the presence of microtubules indicates the co-precipitation of SPD-1 with the microtubules. ( h ) Microtubules were incubated with SPD-1 with or without the R83W mutation and visualized by immunofluorescence following fixation. Scale bar, 20 μm.
Figure Legend Snippet: Physical interaction between SPD-1 and CYK-4 sensitive to SPD-1 R83W mutation. ( a ) In vitro translated full-length CYK-4 was pulled down by full-length SPD-1 immobilized on chitin beads via a chitin-binding domain (CBD) tag. ( b ) In vitro translated full-length SPD-1 was pulled down by full-length CYK-4 or the centralspindlin holocomplex (CYK-4/ZEN-4) immobilized on glutathione-Sepharose beads via a glutathione- S -transferase (GST) tag. ( c ) Schematic drawings of SPD-1 and CYK-4. R83W indicates the mutation found in the spd-1(oj5) mutant exhibiting central spindle defects. ( d ) Yeast 2-hybrid assay of the indicated combinations of bait and prey. Growth on histidine-deficient medium containing 3-amino-1,2,4-triazole (–His+3AT) indicates a positive interaction between the bait and prey. ( e ) SPD-1 1-228 fragment with or without the R83W mutation (WT: wild type) was pulled down by CYK-4 constructs expressed as fusion proteins with maltose-binding protein (MBP) and detected with an anti-SPD-1 antibody. The CYK-4 tail region is necessary and, if dimerized, sufficient for efficient binding. ( f ) The R83W mutation does not affect the mobility of the SPD-1 full-length protein in Superdex 200 size-exclusion chromatography (blue: wild type; red: R83W). The elution profile of a mixture of standard proteins (Thy, thyroglobulin; IgG, gamma globulin; Ova, ovalbumin; MyG, myoglobin; VB12, vitamin B12) is shown in grey. ( g , h ) The R83W mutation does not affect the interaction of SPD-1 with microtubules. ( g ) Wild-type and R83W SPD-1 were incubated with microtubules or control buffer and sedimented by ultracentrifugation. P, pellet; S, supernatant; T, total. Increased recovery in the pellet in the presence of microtubules indicates the co-precipitation of SPD-1 with the microtubules. ( h ) Microtubules were incubated with SPD-1 with or without the R83W mutation and visualized by immunofluorescence following fixation. Scale bar, 20 μm.

Techniques Used: Mutagenesis, In Vitro, Binding Assay, Y2H Assay, Construct, Size-exclusion Chromatography, Incubation, Immunofluorescence

26) Product Images from "IGF-1-induced phosphorylation and altered distribution of TSC1/TSC2 in C2C12 myotubes"

Article Title: IGF-1-induced phosphorylation and altered distribution of TSC1/TSC2 in C2C12 myotubes

Journal: The FEBS journal

doi: 10.1111/j.1742-4658.2010.07635.x

Protein interaction between TSC2 and 14-3-3 protein is mediated thorough phosphorylation of TSC2 at S939 site C2C12 myoblasts were transfected and then differentiated for totally 4 days. A) in vitro GST-14-3-3 pull-down assay. Total cell lysates containing myc-TSC1 (wild type) and Flag-TSC2 (wild-type, S939A or T1462A) proteins were pulled-down with batch-purified GST-14-3-3-beta. Affinity-purified protein complex with Glutathione sepharose beads were then subjected to SDS-PAGE. The interaction between TSC2 mutant S939A and 14-3-3 was clearly decreased compared to wild-type TSC2 or mutant TSC2-T1462A. B) Unphosphorylatable mutant of TSC2 (S939A) showed no increase in the cytosolic relative adundance following the IGF-1 stimulation. Cleared protein lysates were fractionated into the membrane and the cytosolic fraction by ultra-centrifugation. Pan-cadherin antibody was used for the membrane fraction control, and S6K1 was used as cytosolic fraction control. Image-J software (National Institute of Health) was used for the quantification. Protein contents in IGF-1 stimulated sample were quantified as relative amount compared to the simultaneously transfected control. n = 4 per condition. Each value is means ± SE. Amounts of myc-tagged TSC1 and Flag-tagged TSC2 (wild-type) in the cytosolic fraction were increased by IGF-1 stimulation compared to control. In contrast, no changes in the cytosolic pool of myc-TSC1 and Flag-TSC2 (S939A) were observed after IGF-1 stimulation. We could not see significant alterations in the distributions of myc-TSC1 or Flag-TSC2 in the membrane fraction.
Figure Legend Snippet: Protein interaction between TSC2 and 14-3-3 protein is mediated thorough phosphorylation of TSC2 at S939 site C2C12 myoblasts were transfected and then differentiated for totally 4 days. A) in vitro GST-14-3-3 pull-down assay. Total cell lysates containing myc-TSC1 (wild type) and Flag-TSC2 (wild-type, S939A or T1462A) proteins were pulled-down with batch-purified GST-14-3-3-beta. Affinity-purified protein complex with Glutathione sepharose beads were then subjected to SDS-PAGE. The interaction between TSC2 mutant S939A and 14-3-3 was clearly decreased compared to wild-type TSC2 or mutant TSC2-T1462A. B) Unphosphorylatable mutant of TSC2 (S939A) showed no increase in the cytosolic relative adundance following the IGF-1 stimulation. Cleared protein lysates were fractionated into the membrane and the cytosolic fraction by ultra-centrifugation. Pan-cadherin antibody was used for the membrane fraction control, and S6K1 was used as cytosolic fraction control. Image-J software (National Institute of Health) was used for the quantification. Protein contents in IGF-1 stimulated sample were quantified as relative amount compared to the simultaneously transfected control. n = 4 per condition. Each value is means ± SE. Amounts of myc-tagged TSC1 and Flag-tagged TSC2 (wild-type) in the cytosolic fraction were increased by IGF-1 stimulation compared to control. In contrast, no changes in the cytosolic pool of myc-TSC1 and Flag-TSC2 (S939A) were observed after IGF-1 stimulation. We could not see significant alterations in the distributions of myc-TSC1 or Flag-TSC2 in the membrane fraction.

Techniques Used: Transfection, In Vitro, Pull Down Assay, Purification, Affinity Purification, SDS Page, Mutagenesis, Centrifugation, Software

27) Product Images from "Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis"

Article Title: Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis

Journal:

doi: 10.1128/CDLI.12.5.647-653.2005

Expression and purification of K9-K39-K26 chimera. Lane M: molecular weight marker; lane 1: total cell lysate of E. coli expressing GST-K9-K39-K26 fusion protein; lane 2: K9-K39-K26 chimera after Sepharose 4 B purification and cleavage of the GST carrier
Figure Legend Snippet: Expression and purification of K9-K39-K26 chimera. Lane M: molecular weight marker; lane 1: total cell lysate of E. coli expressing GST-K9-K39-K26 fusion protein; lane 2: K9-K39-K26 chimera after Sepharose 4 B purification and cleavage of the GST carrier

Techniques Used: Expressing, Purification, Molecular Weight, Marker

28) Product Images from "TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana"

Article Title: TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1006266

TCS1 physically and genetically interacts with KCBP to control the number of trichome branches. (A) TCS1 interacts with KCBP in yeast cells. (B) TCS1 physically interacts with KCBP in vitro . MBP-TCS1 was pulled down (PD) by GST-KCBP immobilized on Glutathione Sepharose 4B and analyzed by immunoblotting (IB) using an anti-MBP antibody. MBP was used as a negative control. (C) TCS1 interacts with KCBP in vivo . Total proteins from pTCS1 : TCS1-GFP;35SMyc-KCBP and 35S : GFP; 35SMyc-KCBP plants were immunoprecipitated with GFP-Trap-A (IP), and the immunoblots (IB) were probed with anti-GFP and anti-Myc antibodies, respectively. Myc-KCBP was detected in the immunoprecipitated TCS1-GFP complex. (D) Trichome branch (br) distribution of Col-0, tcs1-2 , zwi-101 and zwi-101 tcs1-2 first pair of leaves at 15 days after germination (DAG). Values are given as mean ± SE. (E) The average number of Col-0, tcs1-2 , zwi-101 , zwi-101 tcs1-2 trichome branches treated with (T) or without (UT) 20 μM oryzalin for 2 hours. The branch number of Col-0, tcs1-2 , zwi-101 , zwi-101 tcs1-2 trichomes was examined after a 10-day recovery on ½ MS medium.
Figure Legend Snippet: TCS1 physically and genetically interacts with KCBP to control the number of trichome branches. (A) TCS1 interacts with KCBP in yeast cells. (B) TCS1 physically interacts with KCBP in vitro . MBP-TCS1 was pulled down (PD) by GST-KCBP immobilized on Glutathione Sepharose 4B and analyzed by immunoblotting (IB) using an anti-MBP antibody. MBP was used as a negative control. (C) TCS1 interacts with KCBP in vivo . Total proteins from pTCS1 : TCS1-GFP;35SMyc-KCBP and 35S : GFP; 35SMyc-KCBP plants were immunoprecipitated with GFP-Trap-A (IP), and the immunoblots (IB) were probed with anti-GFP and anti-Myc antibodies, respectively. Myc-KCBP was detected in the immunoprecipitated TCS1-GFP complex. (D) Trichome branch (br) distribution of Col-0, tcs1-2 , zwi-101 and zwi-101 tcs1-2 first pair of leaves at 15 days after germination (DAG). Values are given as mean ± SE. (E) The average number of Col-0, tcs1-2 , zwi-101 , zwi-101 tcs1-2 trichome branches treated with (T) or without (UT) 20 μM oryzalin for 2 hours. The branch number of Col-0, tcs1-2 , zwi-101 , zwi-101 tcs1-2 trichomes was examined after a 10-day recovery on ½ MS medium.

Techniques Used: In Vitro, Negative Control, In Vivo, Immunoprecipitation, Western Blot, Mass Spectrometry

29) Product Images from "Hrs recruits clathrin to early endosomes"

Article Title: Hrs recruits clathrin to early endosomes

Journal: The EMBO Journal

doi: 10.1093/emboj/20.17.5008

Fig. 3. The C-terminus of Hrs binds clathrin TD. ( A ). This illustrates the existence of a potential clathrin-binding motif within residues 770–775 of Hrs. Hrs is the only protein that has the clathrin box motif at the very C-terminus. (B–E) Interaction of Hrs with clathrin. ( B and C ) L40 reporter yeast cells were transformed with bait constructs in pLexA and prey constructs in pGAD. Reporter β-galactosidase activities (in arbitrary units) indicate binding and are represented as mean values of two independent experiments performed in duplicate. Error bars denote ± SEM. In (B), a clathrin triskelion (consisting of three heavy chains) is illustrated, with the terminal domain (TD), distal domain (DD) and hub domain (HD) indicated. ( D ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3) were immobilized on glutathione–Sepharose beads and incubated with pig brain cytosol. The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin with anti-clathrin heavy chain antibodies (upper panel). ( E ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3), were immobilized on glutathione–Sepharose beads and incubated with purified recombinant clathrin terminal domain (TD 1–579 ). The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin-TD 1–579 with anti-clathrin heavy chain (upper panel). Lane 4 represents the total amount of recombinant clathrin-TD 1–579 added to the beads.
Figure Legend Snippet: Fig. 3. The C-terminus of Hrs binds clathrin TD. ( A ). This illustrates the existence of a potential clathrin-binding motif within residues 770–775 of Hrs. Hrs is the only protein that has the clathrin box motif at the very C-terminus. (B–E) Interaction of Hrs with clathrin. ( B and C ) L40 reporter yeast cells were transformed with bait constructs in pLexA and prey constructs in pGAD. Reporter β-galactosidase activities (in arbitrary units) indicate binding and are represented as mean values of two independent experiments performed in duplicate. Error bars denote ± SEM. In (B), a clathrin triskelion (consisting of three heavy chains) is illustrated, with the terminal domain (TD), distal domain (DD) and hub domain (HD) indicated. ( D ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3) were immobilized on glutathione–Sepharose beads and incubated with pig brain cytosol. The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin with anti-clathrin heavy chain antibodies (upper panel). ( E ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3), were immobilized on glutathione–Sepharose beads and incubated with purified recombinant clathrin terminal domain (TD 1–579 ). The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin-TD 1–579 with anti-clathrin heavy chain (upper panel). Lane 4 represents the total amount of recombinant clathrin-TD 1–579 added to the beads.

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

30) Product Images from "Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus"

Article Title: Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024067

ANXA2 specifically binds to pseudoknot RNA. (A) GST pull-down analysis of IBV RNA. Wild-type (WT) and mutant (MT) IBV pseudoknot RNA were labeled with [γ- 32 P] ATP and mixed with GST or GST-ANXA2 protein. Each sample was pulled down with Glutathione 4B Sepharose and pelleted radioactivity was measured with a scintillation counter. Three independent experiments were performed and statistical analysis was done. All experiments were performed in triplicate and mean ± s.d. are shown. ***: p
Figure Legend Snippet: ANXA2 specifically binds to pseudoknot RNA. (A) GST pull-down analysis of IBV RNA. Wild-type (WT) and mutant (MT) IBV pseudoknot RNA were labeled with [γ- 32 P] ATP and mixed with GST or GST-ANXA2 protein. Each sample was pulled down with Glutathione 4B Sepharose and pelleted radioactivity was measured with a scintillation counter. Three independent experiments were performed and statistical analysis was done. All experiments were performed in triplicate and mean ± s.d. are shown. ***: p

Techniques Used: Mutagenesis, Labeling, Radioactivity

31) Product Images from "Jasmonate-Activated MYC2 Represses ETHYLENE INSENSITIVE3 Activity to Antagonize Ethylene-Promoted Apical Hook Formation in Arabidopsis [C] [C] [W]"

Article Title: Jasmonate-Activated MYC2 Represses ETHYLENE INSENSITIVE3 Activity to Antagonize Ethylene-Promoted Apical Hook Formation in Arabidopsis [C] [C] [W]

Journal: The Plant Cell

doi: 10.1105/tpc.113.122002

MYC2 Physically Interacts with EIN3. (A) A pull-down assay shows that EIN3 interacts with MYC2. GST fusion proteins were immobilized with Glutathione Sepharose 4B resin, and then MBP fusion proteins were mixed with the corresponding resin. The precipitated products were separated by 10% SDS-PAGE and further blotted with anti-MBP or anti-GST antibody, respectively. Arrows define the corresponding proteins. (B) A luciferase complementation imaging assay shows that EIN3 and MYC2 interact with each other in N. benthamiana leaves. Agrobacterium strain GV3101 harboring different construct combinations was infiltrated into different N. benthamiana leaf regions. After 3 d of infiltration, luciferase activities were recorded in these regions. cps indicates signal counts per second. (C) . Plant extracts were then immunoprecipitated using anti-MYC antibody, separated on a 10% SDS-PAGE gel, and blotted with anti-MYC or anti-EIN3 antibody. Arrows define the corresponding proteins.
Figure Legend Snippet: MYC2 Physically Interacts with EIN3. (A) A pull-down assay shows that EIN3 interacts with MYC2. GST fusion proteins were immobilized with Glutathione Sepharose 4B resin, and then MBP fusion proteins were mixed with the corresponding resin. The precipitated products were separated by 10% SDS-PAGE and further blotted with anti-MBP or anti-GST antibody, respectively. Arrows define the corresponding proteins. (B) A luciferase complementation imaging assay shows that EIN3 and MYC2 interact with each other in N. benthamiana leaves. Agrobacterium strain GV3101 harboring different construct combinations was infiltrated into different N. benthamiana leaf regions. After 3 d of infiltration, luciferase activities were recorded in these regions. cps indicates signal counts per second. (C) . Plant extracts were then immunoprecipitated using anti-MYC antibody, separated on a 10% SDS-PAGE gel, and blotted with anti-MYC or anti-EIN3 antibody. Arrows define the corresponding proteins.

Techniques Used: Pull Down Assay, SDS Page, Luciferase, Imaging, Construct, Immunoprecipitation

32) Product Images from "The C2A domain in dysferlin is important for association with MG53 (TRIM72)"

Article Title: The C2A domain in dysferlin is important for association with MG53 (TRIM72)

Journal: PLoS Currents

doi: 10.1371/5035add8caff4

COS-7 cells overexpressing FLAG-tagged MG53 were lysed and supplemented with DTT or NEM, and proteins in these lysates were cross-linked with GA. Cross-linked proteins were incubated with glutathione Sepharose 4B beads bound to wild-type C2A-GST, V67D C2A-GST, or GST. GST fusion proteins bound to beads were separated by SDS-PAGE, followed by Coomassie Brilliant Blue R-250-staining. Precipitated MG53 oligomers/monomers were detected on immunoblots using an anti-FLAG antibody. Mutations in the C2A domain affect the association of between dysferlin and MG53.
Figure Legend Snippet: COS-7 cells overexpressing FLAG-tagged MG53 were lysed and supplemented with DTT or NEM, and proteins in these lysates were cross-linked with GA. Cross-linked proteins were incubated with glutathione Sepharose 4B beads bound to wild-type C2A-GST, V67D C2A-GST, or GST. GST fusion proteins bound to beads were separated by SDS-PAGE, followed by Coomassie Brilliant Blue R-250-staining. Precipitated MG53 oligomers/monomers were detected on immunoblots using an anti-FLAG antibody. Mutations in the C2A domain affect the association of between dysferlin and MG53.

Techniques Used: Pull Down Assay, Incubation, SDS Page, Staining, Western Blot

33) Product Images from "Human Papillomavirus Type 8 Interferes with a Novel C/EBP?-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration"

Article Title: Human Papillomavirus Type 8 Interferes with a Novel C/EBP?-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002833

C/EBPβ binds to the enhancer region of CCL20 in vivo. (A) Nucleotide sequence of the human CCL20 promoter region with twelve putative C/EBP binding sites (underlined). Numbers below the underlined C/EBP binding sites mark the sequences, which display C/EBP DNA binding activity in EMSA. In bold is the DNA sequence tested for C/EBP binding in ChIP assay. (B) 32 P-labeled oligonucleotides containing the respective C/EBP binding sites (nt 294–308, nt 574–584, nt 652–667, nt 716–724, nt 734–748) of the CCL20 promoter were incubated with 5 µg GST, GST-C/EBPα or GST-C/EBPβ fusion proteins and analyzed by EMSA. The arrow indicates complexes corresponding to C/EBP DNA binding activity. (C) Chromatin immunoprecipitation assay was performed using RTS3b cells transfected with the C/EBPβ expression vector. For precipitation anti-C/EBPβ (H-7) antibody was used. Genomic DNA was isolated, amplified by real-time PCR with primers specific for the nt 638–677 region of the CCL20 promoter (in bold). The amplicon was quantified (left panel) and visualized on an agarose gel (right panel). The amount of target DNA precipitated with the control antibody was set at 1. Shown are mean values ± SD from four experiments. The asterisk represents statistical significance, p = 0.02.
Figure Legend Snippet: C/EBPβ binds to the enhancer region of CCL20 in vivo. (A) Nucleotide sequence of the human CCL20 promoter region with twelve putative C/EBP binding sites (underlined). Numbers below the underlined C/EBP binding sites mark the sequences, which display C/EBP DNA binding activity in EMSA. In bold is the DNA sequence tested for C/EBP binding in ChIP assay. (B) 32 P-labeled oligonucleotides containing the respective C/EBP binding sites (nt 294–308, nt 574–584, nt 652–667, nt 716–724, nt 734–748) of the CCL20 promoter were incubated with 5 µg GST, GST-C/EBPα or GST-C/EBPβ fusion proteins and analyzed by EMSA. The arrow indicates complexes corresponding to C/EBP DNA binding activity. (C) Chromatin immunoprecipitation assay was performed using RTS3b cells transfected with the C/EBPβ expression vector. For precipitation anti-C/EBPβ (H-7) antibody was used. Genomic DNA was isolated, amplified by real-time PCR with primers specific for the nt 638–677 region of the CCL20 promoter (in bold). The amplicon was quantified (left panel) and visualized on an agarose gel (right panel). The amount of target DNA precipitated with the control antibody was set at 1. Shown are mean values ± SD from four experiments. The asterisk represents statistical significance, p = 0.02.

Techniques Used: In Vivo, Sequencing, Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Labeling, Incubation, Transfection, Expressing, Plasmid Preparation, Isolation, Amplification, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis

HPV8 E7 interferes with binding of C/EBPβ to the CCL20 promoter. (A) Nuclear extracts from HaCaT cells stably expressing HPV8 E7 (pLXSN-HPV8 E7) and corresponding control cells (pLXSN) were analyzed by Western blot for C/EBPβ protein and HMGB1 expression (upper panels). Identical amounts of the respective nuclear extracts were used for EMSA using the 32 P-labeled oligonucleotides (nt 734–748) containing the C/EBP binding site in the CCL20 promoter (lower panel). The complex corresponding to endogenous C/EBP binding activity within the CCL20 promoter is indicated by an arrow. (B) The same cells were used for chromatin immunoprecipitation. Protein-genomic DNA complexes were precipitated with anti-C/EBPβ antibody. DNA was isolated, amplified by real-time PCR with primers specific for the nt 638–677 region of the CCL20 promoter. The amplicon was quantified (lower panel) and visualized on an agarose gel (upper panel). The amount of target DNA precipitated from the pLXSN control cells was set at 100%. The mean values ± SD from three independent experiments are presented. Asterisks represent statistical significance, p = 0.008.
Figure Legend Snippet: HPV8 E7 interferes with binding of C/EBPβ to the CCL20 promoter. (A) Nuclear extracts from HaCaT cells stably expressing HPV8 E7 (pLXSN-HPV8 E7) and corresponding control cells (pLXSN) were analyzed by Western blot for C/EBPβ protein and HMGB1 expression (upper panels). Identical amounts of the respective nuclear extracts were used for EMSA using the 32 P-labeled oligonucleotides (nt 734–748) containing the C/EBP binding site in the CCL20 promoter (lower panel). The complex corresponding to endogenous C/EBP binding activity within the CCL20 promoter is indicated by an arrow. (B) The same cells were used for chromatin immunoprecipitation. Protein-genomic DNA complexes were precipitated with anti-C/EBPβ antibody. DNA was isolated, amplified by real-time PCR with primers specific for the nt 638–677 region of the CCL20 promoter. The amplicon was quantified (lower panel) and visualized on an agarose gel (upper panel). The amount of target DNA precipitated from the pLXSN control cells was set at 100%. The mean values ± SD from three independent experiments are presented. Asterisks represent statistical significance, p = 0.008.

Techniques Used: Binding Assay, Stable Transfection, Expressing, Western Blot, Labeling, Activity Assay, Chromatin Immunoprecipitation, Isolation, Amplification, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis

34) Product Images from "Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis"

Article Title: Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001482

Binding of RNA by dihydrolipoamide acetyltransferases might be a global phenomenon. (A) Hexahistidine-tagged E2 fusion proteins from C. reinhardtii (Cr), S. cerevisiae (Sc), Synechocystis sp. 6803 (Syn), and H. sapiens (Hs) along with two control proteins (PratA and RBP40) were purified on Ni-NTA Sepharose, separated by SDS-PAGE, and Coomassie-stained. To exclude an unspecific RNA binding of contaminating E. coli proteins in (B), we used the same volumes as used for the C. reinhardtii E2 protein of an elution fraction obtained from the bacterial host strain transformed with the empty expression vector served as control (eV). Recombinant proteins are indicated by arrows. Proteins in the preparation of the human E2 subunit (Hs) that are specifically recognized by an anti-histidine antibody are marked by an asterisk. (B) RNA binding assay. One of the 20 (∼100 ng) recombinant proteins shown in (A) was used for UV cross-linking to psbA mRNA. Lanes “ psbA* ” and “ psbA ” show the radiolabeled psbA RNA without and with RNase treatment, respectively. Due to the high intensity of the psbA * signal, a lower exposure of this lane is shown. Specific radioactive signals are indicated by arrows.
Figure Legend Snippet: Binding of RNA by dihydrolipoamide acetyltransferases might be a global phenomenon. (A) Hexahistidine-tagged E2 fusion proteins from C. reinhardtii (Cr), S. cerevisiae (Sc), Synechocystis sp. 6803 (Syn), and H. sapiens (Hs) along with two control proteins (PratA and RBP40) were purified on Ni-NTA Sepharose, separated by SDS-PAGE, and Coomassie-stained. To exclude an unspecific RNA binding of contaminating E. coli proteins in (B), we used the same volumes as used for the C. reinhardtii E2 protein of an elution fraction obtained from the bacterial host strain transformed with the empty expression vector served as control (eV). Recombinant proteins are indicated by arrows. Proteins in the preparation of the human E2 subunit (Hs) that are specifically recognized by an anti-histidine antibody are marked by an asterisk. (B) RNA binding assay. One of the 20 (∼100 ng) recombinant proteins shown in (A) was used for UV cross-linking to psbA mRNA. Lanes “ psbA* ” and “ psbA ” show the radiolabeled psbA RNA without and with RNase treatment, respectively. Due to the high intensity of the psbA * signal, a lower exposure of this lane is shown. Specific radioactive signals are indicated by arrows.

Techniques Used: Binding Assay, Purification, SDS Page, Staining, RNA Binding Assay, Transformation Assay, Expressing, Plasmid Preparation, Recombinant

35) Product Images from "? Subunit of the AP-1 Adaptor Complex Binds Clathrin: Implications for Cooperative Binding in Coated Vesicle Assembly"

Article Title: ? Subunit of the AP-1 Adaptor Complex Binds Clathrin: Implications for Cooperative Binding in Coated Vesicle Assembly

Journal: Molecular Biology of the Cell

doi:

Both LLDLL and LLDLD peptides inhibit GST-LLDLL and GST-LLDLD. Inhibition assays were performed as described under MATERIALS AND METHODS. (A–B) Concentration of each free peptide was 1 mM. (A) GST-LLDLL immobilized on glutathione-Sepharose 4B. (B) Immobilized GST-LLDLD. (C and D) Free peptide concentrations varied from 50 μM to 1 mM. Curves were generated from densitometric analysis of the pellet fractions of the pull-down assays at different peptide concentrations. (C) GST-LLDLL immobilized on glutathione-Sepharose 4B. (D) Immobilized GST-LLDLD.
Figure Legend Snippet: Both LLDLL and LLDLD peptides inhibit GST-LLDLL and GST-LLDLD. Inhibition assays were performed as described under MATERIALS AND METHODS. (A–B) Concentration of each free peptide was 1 mM. (A) GST-LLDLL immobilized on glutathione-Sepharose 4B. (B) Immobilized GST-LLDLD. (C and D) Free peptide concentrations varied from 50 μM to 1 mM. Curves were generated from densitometric analysis of the pellet fractions of the pull-down assays at different peptide concentrations. (C) GST-LLDLL immobilized on glutathione-Sepharose 4B. (D) Immobilized GST-LLDLD.

Techniques Used: Inhibition, Concentration Assay, Generated

36) Product Images from "Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation"

Article Title: Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.9.3103-3115.2003

The catalytic domain of c-Src interacts with PLD2. (A) Schematic representation of GST-fusion protein constructs. (B) c-Src was fragmented into SH3 (88-137), SH2 (144-245), and kinase (265-523) domains. The fragments were cloned as GST fusion proteins, expressed in Escherichia coli , and purified by use of glutathione-conjugated Sepharose beads. An equal amount (2 μg) of GST or GST-Src fragment was incubated with the lysates of COS-7 cells transfected with PLD2 and c-Src. The pull-down proteins were subjected to immunoblot analysis with an antibody against PLD (upper panel). The amount of the GST fusion proteins was visualized by immunoblotting with an anti-GST antibody (lower panel). Data are representative of three experiments.
Figure Legend Snippet: The catalytic domain of c-Src interacts with PLD2. (A) Schematic representation of GST-fusion protein constructs. (B) c-Src was fragmented into SH3 (88-137), SH2 (144-245), and kinase (265-523) domains. The fragments were cloned as GST fusion proteins, expressed in Escherichia coli , and purified by use of glutathione-conjugated Sepharose beads. An equal amount (2 μg) of GST or GST-Src fragment was incubated with the lysates of COS-7 cells transfected with PLD2 and c-Src. The pull-down proteins were subjected to immunoblot analysis with an antibody against PLD (upper panel). The amount of the GST fusion proteins was visualized by immunoblotting with an anti-GST antibody (lower panel). Data are representative of three experiments.

Techniques Used: Construct, Clone Assay, Purification, Incubation, Transfection

37) Product Images from "The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation"

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.776195

Kindlin-2 interacts with K2 C-terminal peptide. Lysates of αIIbβ3-CHO cells transfected with PSGL-1 alone, PSGL-1–K2CT, PSGL-1–K2CTΔ679, or PSGL-1–K2CT double mutant were used for co-IP assays. After incubating with A/G-agarose and PSGL-1 antibody, full-length K2 bound to PSGL-1 constructs was evaluated by SDS-PAGE and Western blotting using anti-K2 (Cell Signaling), which does not detect the C-terminal end of K2. PSGL-1 and K2 levels in total lysates ( TL ) are also shown, with actin used as a loading control.
Figure Legend Snippet: Kindlin-2 interacts with K2 C-terminal peptide. Lysates of αIIbβ3-CHO cells transfected with PSGL-1 alone, PSGL-1–K2CT, PSGL-1–K2CTΔ679, or PSGL-1–K2CT double mutant were used for co-IP assays. After incubating with A/G-agarose and PSGL-1 antibody, full-length K2 bound to PSGL-1 constructs was evaluated by SDS-PAGE and Western blotting using anti-K2 (Cell Signaling), which does not detect the C-terminal end of K2. PSGL-1 and K2 levels in total lysates ( TL ) are also shown, with actin used as a loading control.

Techniques Used: Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Construct, SDS Page, Western Blot

38) Product Images from "H2A.Z Is Required for Global Chromatin Integrity and for Recruitment of RNA Polymerase II under Specific Conditions"

Article Title: H2A.Z Is Required for Global Chromatin Integrity and for Recruitment of RNA Polymerase II under Specific Conditions

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.18.6270-6279.2001

The C-terminal region of H2A.Z interacts with components of the transcriptional machinery. (A) Aligned amino acid sequences of yeast H2A.Z and H2A using BLAST (National Center for Biotechnology Information). Boxed areas represent the C-terminal regions that were fused to GST for the experiments illustrated in panels B and C (red and white) and the M6 region in Drosophila ). (B) GST, GST-H2A (aa 96 to 132), and GST-Z (GST-H2A.Z [aa 103 to 134]) proteins bound to glutathione-Sepharose beads were incubated with a chromatin-enriched yeast extract. L, 2% input of the mixture; S, 2% of the supernatant after pelleting the Sepharose beads; P, washed Sepharose pellet. Samples were analyzed by SDS-PAGE followed by immunoblotting with either an anti-RNA polII antibody or an anti-TBP antibody. (C) The H2A.Z-RNA polII interaction is not mediated by the indirect bridging effect of nucleic acids. The chromatin-enriched extract was treated with or without DNase and RNase and then loaded in a 500-μl glutathione-Sepharose column coupled to GST-H2A.Z (aa 103 to 134). The column was washed and eluted with potassium acetate. L, input of the total reaction; E1 and E2, elutions. Sup-40K is an extract not enriched in chromatin; Pel-40K −DNase is a chromatin-enriched extract not treated with nucleases; Pel-40K +DNase represents the chromatin-enriched extract treated with nucleases. Samples were analyzed as for panel B with an anti-RNA polII antibody.
Figure Legend Snippet: The C-terminal region of H2A.Z interacts with components of the transcriptional machinery. (A) Aligned amino acid sequences of yeast H2A.Z and H2A using BLAST (National Center for Biotechnology Information). Boxed areas represent the C-terminal regions that were fused to GST for the experiments illustrated in panels B and C (red and white) and the M6 region in Drosophila ). (B) GST, GST-H2A (aa 96 to 132), and GST-Z (GST-H2A.Z [aa 103 to 134]) proteins bound to glutathione-Sepharose beads were incubated with a chromatin-enriched yeast extract. L, 2% input of the mixture; S, 2% of the supernatant after pelleting the Sepharose beads; P, washed Sepharose pellet. Samples were analyzed by SDS-PAGE followed by immunoblotting with either an anti-RNA polII antibody or an anti-TBP antibody. (C) The H2A.Z-RNA polII interaction is not mediated by the indirect bridging effect of nucleic acids. The chromatin-enriched extract was treated with or without DNase and RNase and then loaded in a 500-μl glutathione-Sepharose column coupled to GST-H2A.Z (aa 103 to 134). The column was washed and eluted with potassium acetate. L, input of the total reaction; E1 and E2, elutions. Sup-40K is an extract not enriched in chromatin; Pel-40K −DNase is a chromatin-enriched extract not treated with nucleases; Pel-40K +DNase represents the chromatin-enriched extract treated with nucleases. Samples were analyzed as for panel B with an anti-RNA polII antibody.

Techniques Used: Incubation, SDS Page

39) Product Images from "The Leber Congenital Amaurosis Protein AIPL1 Functions as Part of a Chaperone Heterocomplex"

Article Title: The Leber Congenital Amaurosis Protein AIPL1 Functions as Part of a Chaperone Heterocomplex

Journal: Investigative ophthalmology & visual science

doi: 10.1167/iovs.07-1576

Characterization of AIPL1-chaperone interactions using in vitro protein-binding assays. ( A ) Affinity pull-down of endogenous Hsp90 and Hsp70 with GST-AIPL1. GST and GST-AIPL1 were affinity purified on glutathione Sepharose 4B ( lower ; Coomassie stain).
Figure Legend Snippet: Characterization of AIPL1-chaperone interactions using in vitro protein-binding assays. ( A ) Affinity pull-down of endogenous Hsp90 and Hsp70 with GST-AIPL1. GST and GST-AIPL1 were affinity purified on glutathione Sepharose 4B ( lower ; Coomassie stain).

Techniques Used: In Vitro, Protein Binding, Affinity Purification, Staining

40) Product Images from "Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity"

Article Title: Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity

Journal: eLife

doi: 10.7554/eLife.11058

Arginine contributes to the regulation of Rheb activity by TSC2. ( A ) Model of the Rheb-TSC2 GAP domain complex, built using the human Rap1-Rap1GAP complex (pdb:3BRW) as a template. The TSC2 is in cyan cartoon representation, the Rheb is in magenta cartoon representation with a transparent molecular surface shown. The bound GTP is shown in spacefilling representation and the four key mutated Rheb residues, aspartic acid (Asp) 60, glutamine (Gln) 64, asparagine (Asn) 119, and asparagine (Asn) 153, are shown in stick representation. Note that these residues are not likely to perturb the interaction between Rheb and TSC2 directly. In addition, the location of the C-terminal membrane anchor of Rheb is shown. The figure was generated with PyMOL (Schrodinger, LLC). ( B ) Rheb R15G mutant is GTP-bound and largely insensitive to TSC2-dependent hydrolysis. Rheb wild-type and Rheb R15G were loaded with radiolabeled GTP prior to incubation with TSC2 for 60 min. GTP and GDP were eluted from Rheb and analyzed by thin-layer chromatography. Immunoblot (IB) samples demonstrate protein loading controls for Rheb, TSC1 and TSC2. ( C ) HeLa cells were transfected with Flag-tagged wild-type Rheb, active Rheb N153T, or inactive Rheb N119I for 24 hr. Cells (in full nutrient conditions) were fixed and immunostained with antibodies against TSC2 and Flag-tagged Rheb. ( D ) HeLa cells were transfected with Flag-tagged wild-type Rheb, active Rheb R15G, or inactive Rheb D60K for 24 hr. Cells were starved of amino acids and serum prior to fixation. Cells were immunostained with antibodies against TSC2 and Flag-tagged Rheb. ( E ) HeLa cells were transfected with active and inactive constructs described in C and D. The percentage of cells with punctate TSC2 was quantified in cells expressing Rheb mutants maintained in full nutrient conditions. ( F, G ) Amino acids, specifically arginine regulate the interaction between Rheb and mTOR. HeLa cells were transfected with Flag-wild-type Rheb overnight, lysed and loaded onto anti-Flag beads. HeLa cells transfected with myc-mTOR were subject to starvation protocols as indicated prior to lysis. Lysates were incubated with Rheb-loaded beads at 4°C. Samples were analyzed by immunoblotting to detect interaction of mTOR with Rheb. ( H ) TSC complex can inhibit Rheb-mediated mTORC1 activity independent of GAP activity. Cells were transfected with Flag-wild-type Rheb, Flag-Rheb R15G or Flag-Rheb N153T with Flag-TSC2 and HA-GST-tagged S6K overnight as in Figure 4H . Cells were subjected to arginine starvation in the presence of dFCS, prior to lysis and immunoblotted for phosphorylation of S6K. ( I ) HeLa cells were transfected overnight with wild-type or GAP-deficient TSC2 and HA-GST-tagged S6K. Cells were lysed and immunoblotted for phosphorylation of S6K. ( J ) HeLa cells were transfected with GST-Rheb either in the presence of mTOR and TSC2 constructs as indicated. Lysates were subjected to pull-down with glutathione Sepharose beads and interaction between Rheb and mTOR was analyzed by immunoblot. All graphs represent an average of at least two independent experiments and error bars represent standard deviation. Scale bars: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.11058.014
Figure Legend Snippet: Arginine contributes to the regulation of Rheb activity by TSC2. ( A ) Model of the Rheb-TSC2 GAP domain complex, built using the human Rap1-Rap1GAP complex (pdb:3BRW) as a template. The TSC2 is in cyan cartoon representation, the Rheb is in magenta cartoon representation with a transparent molecular surface shown. The bound GTP is shown in spacefilling representation and the four key mutated Rheb residues, aspartic acid (Asp) 60, glutamine (Gln) 64, asparagine (Asn) 119, and asparagine (Asn) 153, are shown in stick representation. Note that these residues are not likely to perturb the interaction between Rheb and TSC2 directly. In addition, the location of the C-terminal membrane anchor of Rheb is shown. The figure was generated with PyMOL (Schrodinger, LLC). ( B ) Rheb R15G mutant is GTP-bound and largely insensitive to TSC2-dependent hydrolysis. Rheb wild-type and Rheb R15G were loaded with radiolabeled GTP prior to incubation with TSC2 for 60 min. GTP and GDP were eluted from Rheb and analyzed by thin-layer chromatography. Immunoblot (IB) samples demonstrate protein loading controls for Rheb, TSC1 and TSC2. ( C ) HeLa cells were transfected with Flag-tagged wild-type Rheb, active Rheb N153T, or inactive Rheb N119I for 24 hr. Cells (in full nutrient conditions) were fixed and immunostained with antibodies against TSC2 and Flag-tagged Rheb. ( D ) HeLa cells were transfected with Flag-tagged wild-type Rheb, active Rheb R15G, or inactive Rheb D60K for 24 hr. Cells were starved of amino acids and serum prior to fixation. Cells were immunostained with antibodies against TSC2 and Flag-tagged Rheb. ( E ) HeLa cells were transfected with active and inactive constructs described in C and D. The percentage of cells with punctate TSC2 was quantified in cells expressing Rheb mutants maintained in full nutrient conditions. ( F, G ) Amino acids, specifically arginine regulate the interaction between Rheb and mTOR. HeLa cells were transfected with Flag-wild-type Rheb overnight, lysed and loaded onto anti-Flag beads. HeLa cells transfected with myc-mTOR were subject to starvation protocols as indicated prior to lysis. Lysates were incubated with Rheb-loaded beads at 4°C. Samples were analyzed by immunoblotting to detect interaction of mTOR with Rheb. ( H ) TSC complex can inhibit Rheb-mediated mTORC1 activity independent of GAP activity. Cells were transfected with Flag-wild-type Rheb, Flag-Rheb R15G or Flag-Rheb N153T with Flag-TSC2 and HA-GST-tagged S6K overnight as in Figure 4H . Cells were subjected to arginine starvation in the presence of dFCS, prior to lysis and immunoblotted for phosphorylation of S6K. ( I ) HeLa cells were transfected overnight with wild-type or GAP-deficient TSC2 and HA-GST-tagged S6K. Cells were lysed and immunoblotted for phosphorylation of S6K. ( J ) HeLa cells were transfected with GST-Rheb either in the presence of mTOR and TSC2 constructs as indicated. Lysates were subjected to pull-down with glutathione Sepharose beads and interaction between Rheb and mTOR was analyzed by immunoblot. All graphs represent an average of at least two independent experiments and error bars represent standard deviation. Scale bars: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.11058.014

Techniques Used: Activity Assay, Generated, Mutagenesis, Incubation, Thin Layer Chromatography, Transfection, Construct, Expressing, Lysis, Standard Deviation

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    GE Healthcare glutathione sepharose 4b
    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on <t>glutathione-sepharose</t> beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
    Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 2034 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
    Glutathione Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

    doi: 10.1016/j.omtn.2018.02.011

    Figure Lengend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Article Snippet: Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA).

    Techniques: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

    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.

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M806152200

    Figure Lengend 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.

    Article Snippet: After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ).

    Techniques: 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.

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M806152200

    Figure Lengend 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.

    Article Snippet: After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ).

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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0025694

    Figure Lengend 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.

    Article Snippet: After the incubation, the glutathione-Sepharose was washed three times with B400 buffer, washed two times with B100 buffer, and then incubated with nuclear extracts from HeLa cells at 4°C for 2 hr.

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0025694

    Figure Lengend 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.

    Article Snippet: After the incubation, the glutathione-Sepharose was washed three times with B400 buffer, washed two times with B100 buffer, and then incubated with nuclear extracts from HeLa cells at 4°C for 2 hr.

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

    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Journal: International Journal of Molecular Sciences

    Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

    doi: 10.3390/ijms15022929

    Figure Lengend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Article Snippet: The soluble fraction of the lysed cells was incubated 2 h at 4 °C with 10 μg GST alone or GST-tagged golgin-160(1–393) that had been pre-conjugated to glutathione-Sepharose 4B beads.

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