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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Images

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 "STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer"

Article Title: STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0808-1

STK25 interacts with GOLPH3 and regulates its expression. a , b Exogenous STK25 interacts with GOLPH3. Cells were transfected with the indicated plasmids. Co-IP was performed using FLAG antibody to pull down FLAG-STK25 ( a ) or anti-Myc against Myc-GOLPH3 ( b ). Then, STK25 and GOLPH3 were detected with the indicated antibodies. c , d His-STK25 interacts directly with GST-GOLPH3 but not GST by in vitro GST pull-down and His-tag pull-down assays, respectively. e STK25 overexpression decreases GLOPH3 mRNA and protein levels in CRC cells. f STK25 knockdown increases GOLPH3 mRNA and protein levels in CRC cells
Figure Legend Snippet: STK25 interacts with GOLPH3 and regulates its expression. a , b Exogenous STK25 interacts with GOLPH3. Cells were transfected with the indicated plasmids. Co-IP was performed using FLAG antibody to pull down FLAG-STK25 ( a ) or anti-Myc against Myc-GOLPH3 ( b ). Then, STK25 and GOLPH3 were detected with the indicated antibodies. c , d His-STK25 interacts directly with GST-GOLPH3 but not GST by in vitro GST pull-down and His-tag pull-down assays, respectively. e STK25 overexpression decreases GLOPH3 mRNA and protein levels in CRC cells. f STK25 knockdown increases GOLPH3 mRNA and protein levels in CRC cells

Techniques Used: Expressing, Transfection, Co-Immunoprecipitation Assay, In Vitro, Over Expression

3) Product Images from "PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)"

Article Title: PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)

Journal: Scientific Reports

doi: 10.1038/s41598-019-42366-9

PfMSA180 (PF3D7_1014100) exists as a 170 kDa protein in parasites. ( A ) Schematic representation of full-length PfMSA180 and recombinant protein fragments (Tr 1–5) PfMSA180 consists of 1455 aa with a calculated molecular mass (MW) of 173.3 kDa. The protein has a predicted signal peptide (SP; 1 to 22 aa; shown in green). Recombinant PfMSA180 truncates were expressed as N-terminal GST-tagged proteins by the wheat germ cell-free system (WGCFS). Tr1, residues E 22 -S 263 (expected MW 29 kDa); Tr2, A 264 -D 501 (expected MW 27.9 kDa); Tr3, I 508 -P 723 (expected MW 52.8 kDa, including the GST tag); Tr4, A 805 -P 1093 (expected MW 34 0.4 kDa); and Tr5, L 1193 -P 1455 (expected MW 58.5 kDa including the GST tag). ( B ) Recombinant PfMSA180 truncates are shown stained with Coomassie brilliant blue (CBB) following purification on a glutathione-Sepharose 4B column and resolution by 12.5% SDS-PAGE under reducing conditions. Arrowheads indicate molecular masses predicted from amino acid sequences of the corresponding recombinant PfMSA180 truncates (original data is included in the Supplementary Data File 1 ). ( C ) Reactivity of rabbit anti-PfMSA180 antibodies to native PfMSA180. Schizont-rich parasite pellets were solubilized with NP40 and PfMSA180 immunoprecipitated with the indicated mouse antibodies. Mouse anti-HisGST antibody was used as a negative control. Immunoprecipitated full length PfMSA180 was detected using rabbit antibody to each of the truncates; indicated using an arrowhead. The additional bands observed in the membrane fraction at 120, 80, and 45 kDa, were likely products of protein SUB1-mediated proteolysis of full-length protein. Antibodies to truncate 3 did not immunoprecipitate protein (original data is included in the Supplementary Data File 1 ).
Figure Legend Snippet: PfMSA180 (PF3D7_1014100) exists as a 170 kDa protein in parasites. ( A ) Schematic representation of full-length PfMSA180 and recombinant protein fragments (Tr 1–5) PfMSA180 consists of 1455 aa with a calculated molecular mass (MW) of 173.3 kDa. The protein has a predicted signal peptide (SP; 1 to 22 aa; shown in green). Recombinant PfMSA180 truncates were expressed as N-terminal GST-tagged proteins by the wheat germ cell-free system (WGCFS). Tr1, residues E 22 -S 263 (expected MW 29 kDa); Tr2, A 264 -D 501 (expected MW 27.9 kDa); Tr3, I 508 -P 723 (expected MW 52.8 kDa, including the GST tag); Tr4, A 805 -P 1093 (expected MW 34 0.4 kDa); and Tr5, L 1193 -P 1455 (expected MW 58.5 kDa including the GST tag). ( B ) Recombinant PfMSA180 truncates are shown stained with Coomassie brilliant blue (CBB) following purification on a glutathione-Sepharose 4B column and resolution by 12.5% SDS-PAGE under reducing conditions. Arrowheads indicate molecular masses predicted from amino acid sequences of the corresponding recombinant PfMSA180 truncates (original data is included in the Supplementary Data File 1 ). ( C ) Reactivity of rabbit anti-PfMSA180 antibodies to native PfMSA180. Schizont-rich parasite pellets were solubilized with NP40 and PfMSA180 immunoprecipitated with the indicated mouse antibodies. Mouse anti-HisGST antibody was used as a negative control. Immunoprecipitated full length PfMSA180 was detected using rabbit antibody to each of the truncates; indicated using an arrowhead. The additional bands observed in the membrane fraction at 120, 80, and 45 kDa, were likely products of protein SUB1-mediated proteolysis of full-length protein. Antibodies to truncate 3 did not immunoprecipitate protein (original data is included in the Supplementary Data File 1 ).

Techniques Used: Recombinant, Staining, Purification, SDS Page, Immunoprecipitation, Negative Control

PfMSA180 is expressed in schizonts and localizes on the merozoite surface. ( A ) A time course immunoblot analysis of 3D7 parasite extracts shows that PfMSA180 is synthesized late in the intraerythrocytic cycle. The protein extracts prepared from Percoll-sorbitol-synchronized parasites were electrophoresed on SDS-12.5% PAGE and probed with rabbit polyclonal anti-PfMSA180 Tr1 antibodies. Hours post-invasion (hpi) are indicated for each lane. Anti-PfHSP70 monoclonal antibody was used as a quantitative parasite protein marker, anti-human spectrin α I rabbit antibody (Santa Cruz Biotechnology, Dallas, TX) indicating the number of loaded erythrocytes 49 , and anti-AMA1 antibody was used as a mature schizont stage marker (original data is included in the Supplementary Data File 1 ). ( B ) IFA analysis of PfMSA180 with MTIP (myosin A tail domain interacting protein). Free merozoites were processed with (+) or without (−) permeabilization using 0.1% Triton X-100 (TrX-100). The merozoites were stained with anti-PfMSA180-Tr1 antibody (upper panels, green color) or anti-PfMSA180-Tr4 (lower panels, green color) and co-stained with anti-MTIP antibody (red color). The leftmost panels show pictures of differential interference contrast (DIC). The rightmost panels show merged pictures with DAPI, showing localization of parasite’s nucleus. ( C ) IFA analysis of PfMSA180 with AMA1 and MSP1-19. Free merozoites were permeabilized with 0.1% Triton X-100. The merozoites were stained with anti-PfMSA180-Tr1 antibody (upper panels, green color) or anti-PfMSA180-Tr4 (lower panels, green color) and co-stained with anti-AMA1 antibody or anti-MSP1-19 antibody (red color). The leftmost panels show pictures of differential interference contrast (DIC). The rightmost panels show merged pictures with DAPI, showing localization of parasite’s nucleus. ( D ) Fractionation of PfMSA180 due to solubility. Schizont-rich 3D7 parasites were disrupted by sonication and fractionated with ultracentrifugation. The resulting soluble and membrane fractions were applied to SDS-PAGE. Immunoblotting analysis with anti-Tr1 antibody detected PfMSA180 in the membrane fraction. MSP3, a secreted protein was detected in both membrane and soluble fractions. AMA1, an integral membrane protein, and MSP1-19, a peripheral GPI-anchored membrane protein, were detected in the membrane fraction. TL: total schizont-rich parasite lysate; S: soluble fractions; M: membrane fraction. Original data is included in the Supplementary Data File 1 .
Figure Legend Snippet: PfMSA180 is expressed in schizonts and localizes on the merozoite surface. ( A ) A time course immunoblot analysis of 3D7 parasite extracts shows that PfMSA180 is synthesized late in the intraerythrocytic cycle. The protein extracts prepared from Percoll-sorbitol-synchronized parasites were electrophoresed on SDS-12.5% PAGE and probed with rabbit polyclonal anti-PfMSA180 Tr1 antibodies. Hours post-invasion (hpi) are indicated for each lane. Anti-PfHSP70 monoclonal antibody was used as a quantitative parasite protein marker, anti-human spectrin α I rabbit antibody (Santa Cruz Biotechnology, Dallas, TX) indicating the number of loaded erythrocytes 49 , and anti-AMA1 antibody was used as a mature schizont stage marker (original data is included in the Supplementary Data File 1 ). ( B ) IFA analysis of PfMSA180 with MTIP (myosin A tail domain interacting protein). Free merozoites were processed with (+) or without (−) permeabilization using 0.1% Triton X-100 (TrX-100). The merozoites were stained with anti-PfMSA180-Tr1 antibody (upper panels, green color) or anti-PfMSA180-Tr4 (lower panels, green color) and co-stained with anti-MTIP antibody (red color). The leftmost panels show pictures of differential interference contrast (DIC). The rightmost panels show merged pictures with DAPI, showing localization of parasite’s nucleus. ( C ) IFA analysis of PfMSA180 with AMA1 and MSP1-19. Free merozoites were permeabilized with 0.1% Triton X-100. The merozoites were stained with anti-PfMSA180-Tr1 antibody (upper panels, green color) or anti-PfMSA180-Tr4 (lower panels, green color) and co-stained with anti-AMA1 antibody or anti-MSP1-19 antibody (red color). The leftmost panels show pictures of differential interference contrast (DIC). The rightmost panels show merged pictures with DAPI, showing localization of parasite’s nucleus. ( D ) Fractionation of PfMSA180 due to solubility. Schizont-rich 3D7 parasites were disrupted by sonication and fractionated with ultracentrifugation. The resulting soluble and membrane fractions were applied to SDS-PAGE. Immunoblotting analysis with anti-Tr1 antibody detected PfMSA180 in the membrane fraction. MSP3, a secreted protein was detected in both membrane and soluble fractions. AMA1, an integral membrane protein, and MSP1-19, a peripheral GPI-anchored membrane protein, were detected in the membrane fraction. TL: total schizont-rich parasite lysate; S: soluble fractions; M: membrane fraction. Original data is included in the Supplementary Data File 1 .

Techniques Used: Synthesized, Polyacrylamide Gel Electrophoresis, Marker, Immunofluorescence, Staining, Fractionation, Solubility, Sonication, SDS Page

4) Product Images from "Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4"

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4

Journal: Scientific Reports

doi: 10.1038/s41598-018-38134-w

Introduction of mutations in predicted Can f 6 epitopes. ( A ) Highly conserved region among Can f 6, Fel d 4, and Equ c 1—but not among other representative lipocalin allergens (indicated by a green bar above the sequences)—was predicted to contain IgE epitope(s) involved in cross-reactivity. Three sites composed of three successive amino acids containing charged residues were substituted with triple alanine, and were designated as rCan f 6-mu-1, mu-2, and mu-3. ( B ) Schematic representation of the mutation sites (shown in red) in a ribbon diagram (left) and surface model (right) of rCan f 6.
Figure Legend Snippet: Introduction of mutations in predicted Can f 6 epitopes. ( A ) Highly conserved region among Can f 6, Fel d 4, and Equ c 1—but not among other representative lipocalin allergens (indicated by a green bar above the sequences)—was predicted to contain IgE epitope(s) involved in cross-reactivity. Three sites composed of three successive amino acids containing charged residues were substituted with triple alanine, and were designated as rCan f 6-mu-1, mu-2, and mu-3. ( B ) Schematic representation of the mutation sites (shown in red) in a ribbon diagram (left) and surface model (right) of rCan f 6.

Techniques Used: Mutagenesis

X-ray crystal structure of Can f 6 with characteristics typical of lipocalin-like proteins. ( A ) Tertiary structure of rCan f 6 (chain A) represented in a ribbon diagram. A β-barrel structure composed of 8 β-strands (indicated by A–H) is shown in green, while an α-helix and 3 10 -helix structures are indicated in orange. Yellow sticks represent an intramolecular disulfide bond. ( B ) Positions of typical secondary structures in rCan f 6 (chain A) are represented along with its amino acid sequence.
Figure Legend Snippet: X-ray crystal structure of Can f 6 with characteristics typical of lipocalin-like proteins. ( A ) Tertiary structure of rCan f 6 (chain A) represented in a ribbon diagram. A β-barrel structure composed of 8 β-strands (indicated by A–H) is shown in green, while an α-helix and 3 10 -helix structures are indicated in orange. Yellow sticks represent an intramolecular disulfide bond. ( B ) Positions of typical secondary structures in rCan f 6 (chain A) are represented along with its amino acid sequence.

Techniques Used: Sequencing

Effect of predicted Can f 6 epitope mutations on IgE reactivity. ( A ) Relative IgE reactivity to the mutated rCan f 6 proteins compared with rCan f 6 was evaluated by ELISA. Can f 6-reactive sera from 18 patients were subjected to this assay. Lines in individual columns denote the median. ( B ) Inhibition ELISA to assess specific IgE binding to immobilized rCan f 6 in the presense of wild-type and mutated rCan f 6 proteins as competitors. Sera from patients 16, 25 and 35 were used for this assay. ( C ) IgE-western blotting of Can f 6. rCan f 6 and mutated rCan f 6 proteins were subjected to SDS-PAGE and then transferred to PVDF membranes. The blots were treated with the serum from the 3 patients (upper panels). The blots were also stained with Ponceau S to verify proper protein transfer (lower panels).
Figure Legend Snippet: Effect of predicted Can f 6 epitope mutations on IgE reactivity. ( A ) Relative IgE reactivity to the mutated rCan f 6 proteins compared with rCan f 6 was evaluated by ELISA. Can f 6-reactive sera from 18 patients were subjected to this assay. Lines in individual columns denote the median. ( B ) Inhibition ELISA to assess specific IgE binding to immobilized rCan f 6 in the presense of wild-type and mutated rCan f 6 proteins as competitors. Sera from patients 16, 25 and 35 were used for this assay. ( C ) IgE-western blotting of Can f 6. rCan f 6 and mutated rCan f 6 proteins were subjected to SDS-PAGE and then transferred to PVDF membranes. The blots were treated with the serum from the 3 patients (upper panels). The blots were also stained with Ponceau S to verify proper protein transfer (lower panels).

Techniques Used: Enzyme-linked Immunosorbent Assay, Inhibition, Binding Assay, Western Blot, SDS Page, Staining

Purification of rCan f 6. ( A ) Gel filtration chromatogram of the purified rCan f 6. ( B , C ) SDS-PAGE profiles of rCan f 6. Purified recombinant protein (3 µg/lane) was electrophoretically separated under ( B ) reducing or ( C ) non-reducing conditions and then stained with Coomassie Brilliant blue. ( D ) Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra of rCan f 6. Mass spectrometry of the purified recombinant protein was carried out in the linear mode using sinapinic acid as a matrix. The  m/z  value of the main peak (20337.47) corresponds to the deduced molecular mass of the recombinant protein. The sub-peak ( m/z  20550) is considered to be derived from rCan f 6 complexed with sinapinic acid. ( E ) Distribution states of rCan f 6 analysed by AUC-SV. The molecular mass of rCan f 6 was calculated as 19.9 kDa.
Figure Legend Snippet: Purification of rCan f 6. ( A ) Gel filtration chromatogram of the purified rCan f 6. ( B , C ) SDS-PAGE profiles of rCan f 6. Purified recombinant protein (3 µg/lane) was electrophoretically separated under ( B ) reducing or ( C ) non-reducing conditions and then stained with Coomassie Brilliant blue. ( D ) Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra of rCan f 6. Mass spectrometry of the purified recombinant protein was carried out in the linear mode using sinapinic acid as a matrix. The m/z value of the main peak (20337.47) corresponds to the deduced molecular mass of the recombinant protein. The sub-peak ( m/z 20550) is considered to be derived from rCan f 6 complexed with sinapinic acid. ( E ) Distribution states of rCan f 6 analysed by AUC-SV. The molecular mass of rCan f 6 was calculated as 19.9 kDa.

Techniques Used: Purification, Filtration, SDS Page, Recombinant, Staining, Mass Spectrometry, Derivative Assay

Analysis of IgE cross-reactivity between Can f 6 and Fel d 4. Inhibition ELISA to assess specific IgE binding to immobilized rFel d 4 in the presence of rFel d 4, wild-type and mutated rCan f 6 proteins as competitors. Sera from patients 16, 25 and 35 were used for this assay.
Figure Legend Snippet: Analysis of IgE cross-reactivity between Can f 6 and Fel d 4. Inhibition ELISA to assess specific IgE binding to immobilized rFel d 4 in the presence of rFel d 4, wild-type and mutated rCan f 6 proteins as competitors. Sera from patients 16, 25 and 35 were used for this assay.

Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay

5) Product Images from "Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion"

Article Title: Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion

Journal: Nature Communications

doi: 10.1038/s41467-018-06906-7

ILK L207W mutation disrupts the F-actin bundling. a (Top left) Structural comparison of the ILK KLD bound to α-Parvin CH2 and (colored in gray; PDB ID 3KMW) and its comparison with the ILK mutant form (colored in blue). The superposition of the mutant ILK KLD (267 aligned Cα atoms) to the ATP-bound (PDB ID 3KMW) and -free (PDB ID 3KMU) forms shows overall similarities with root-mean-square deviations of 0.58 Å and 0.47 Å, respectively. A small conformational change is observed in the ATP-binding site of the mutant ILK KLD likely due to mutation or distinct crystal packing. (Top right) Close-up view of the ATP-binding sites of the ILK KLD between ATP-bound wild type (gray) and deficient mutant (blue). Mg, ATP, L207 in the wild type, and W207 in the mutant ILK KLDs are highlighted in ball and stick models. (Bottom) Close-up stereo view of the loss-of-ATP-binding mutation site in the ILK KLD. The 2Fo-Fc electron density map contoured at 1 σ is shown in gray mesh. The Fo–Fc omit map calculated from the mutant structure without the residue (W207), contoured at 3.5 σ, is overlaid (red mesh). Selected residues in the ATP-binding site are labeled. b Representative microscopic image showing that IPP L207W impaired F-actin bundle formation (no larger bundles) as compared with the WT IPP in ( c ). Selected microscopic image showing F-actin bundles in the presence of WT IPP. Bar = 100 μm. d Quantitative comparison of the F-actin bundle sizes of the randomly selected 20 slides showing the mutation dramatically reduced the F-actin bundle sizes (red squares) as compared with those induced by WT IPP (blue triangles)
Figure Legend Snippet: ILK L207W mutation disrupts the F-actin bundling. a (Top left) Structural comparison of the ILK KLD bound to α-Parvin CH2 and (colored in gray; PDB ID 3KMW) and its comparison with the ILK mutant form (colored in blue). The superposition of the mutant ILK KLD (267 aligned Cα atoms) to the ATP-bound (PDB ID 3KMW) and -free (PDB ID 3KMU) forms shows overall similarities with root-mean-square deviations of 0.58 Å and 0.47 Å, respectively. A small conformational change is observed in the ATP-binding site of the mutant ILK KLD likely due to mutation or distinct crystal packing. (Top right) Close-up view of the ATP-binding sites of the ILK KLD between ATP-bound wild type (gray) and deficient mutant (blue). Mg, ATP, L207 in the wild type, and W207 in the mutant ILK KLDs are highlighted in ball and stick models. (Bottom) Close-up stereo view of the loss-of-ATP-binding mutation site in the ILK KLD. The 2Fo-Fc electron density map contoured at 1 σ is shown in gray mesh. The Fo–Fc omit map calculated from the mutant structure without the residue (W207), contoured at 3.5 σ, is overlaid (red mesh). Selected residues in the ATP-binding site are labeled. b Representative microscopic image showing that IPP L207W impaired F-actin bundle formation (no larger bundles) as compared with the WT IPP in ( c ). Selected microscopic image showing F-actin bundles in the presence of WT IPP. Bar = 100 μm. d Quantitative comparison of the F-actin bundle sizes of the randomly selected 20 slides showing the mutation dramatically reduced the F-actin bundle sizes (red squares) as compared with those induced by WT IPP (blue triangles)

Techniques Used: Mutagenesis, Binding Assay, Labeling

IPP interaction with F-actin. a Schematic organization of IPP based on structural data. ILK binds to PINCH LIM1 via its ankyrin domain and α-Parvin CH2 via its pseudokinase domain, respectively. The Wiscott–Aldrich syndrome protein (WASP) homology domain (WH2) motifs are highlighted in PINCH and α-Parvin. b A representative gel filtration profile of the purified IPP complex by Superose 6 10/300 GL size exclusion chromatography column (GE healthcare). The eluted peak is overlaid with an elution curve of standard molecular weight proteins (dot lines). c Co-sedimentation of IPP at dose-dependent amounts in the presence/absence of F-actin. The F-actin was incubated at 2.3 μM constant concentration with increasing concentrations of each test sample in 5% glycerol containing protein buffer. Representative gels with Coomassie stain are shown. M marker proteins, S supernatant, P pellets
Figure Legend Snippet: IPP interaction with F-actin. a Schematic organization of IPP based on structural data. ILK binds to PINCH LIM1 via its ankyrin domain and α-Parvin CH2 via its pseudokinase domain, respectively. The Wiscott–Aldrich syndrome protein (WASP) homology domain (WH2) motifs are highlighted in PINCH and α-Parvin. b A representative gel filtration profile of the purified IPP complex by Superose 6 10/300 GL size exclusion chromatography column (GE healthcare). The eluted peak is overlaid with an elution curve of standard molecular weight proteins (dot lines). c Co-sedimentation of IPP at dose-dependent amounts in the presence/absence of F-actin. The F-actin was incubated at 2.3 μM constant concentration with increasing concentrations of each test sample in 5% glycerol containing protein buffer. Representative gels with Coomassie stain are shown. M marker proteins, S supernatant, P pellets

Techniques Used: Filtration, Purification, Size-exclusion Chromatography, Molecular Weight, Sedimentation, Incubation, Concentration Assay, Staining, Marker

Identification of actin-binding WH2 motifs in PINCH-1 and α-Parvin. a Sequence alignment of PINCH-1 C-terminal tail and α--terminus with representative WH2 motifs found in other proteins showing the presence of distinct WH2 motifs in PINCH and Parvin. b 0.1 mM 1 H- 15 N HSQC of α-Parvin-N in the absence and presence of 0.2 mM AP-actin showing that α-Parvin-N has potent binding to G-actin (left panel). Mutation of putative G-actin-binding residues L37A/R39A/R40A/K41A/K42A (α-Parvin 5 A) drastically reduces actin binding (right panel). c Co-sedimentation assay showing α-Parvin-N binds to F-actin potently. +, ++, +++ correspond to the concentration of α-Parvin-N at 7.7 μM, 23.0 μM, and 76.7 μM, respectively
Figure Legend Snippet: Identification of actin-binding WH2 motifs in PINCH-1 and α-Parvin. a Sequence alignment of PINCH-1 C-terminal tail and α--terminus with representative WH2 motifs found in other proteins showing the presence of distinct WH2 motifs in PINCH and Parvin. b 0.1 mM 1 H- 15 N HSQC of α-Parvin-N in the absence and presence of 0.2 mM AP-actin showing that α-Parvin-N has potent binding to G-actin (left panel). Mutation of putative G-actin-binding residues L37A/R39A/R40A/K41A/K42A (α-Parvin 5 A) drastically reduces actin binding (right panel). c Co-sedimentation assay showing α-Parvin-N binds to F-actin potently. +, ++, +++ correspond to the concentration of α-Parvin-N at 7.7 μM, 23.0 μM, and 76.7 μM, respectively

Techniques Used: Binding Assay, Sequencing, Mutagenesis, Sedimentation, Concentration Assay

6) Product Images from "The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation"

Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.9.3577-3587.2004

Effect of PABP and Apc5 on translation in vitro. (A) Krebs-2 cell-free translation reaction mixtures that had been pretreated with GST-Paip2 for PABP depletion or with GST as a control were programmed with 100 ng of the indicated transcripts at 37°C for 90 min followed by measurements of firefly luciferase activity. Immunoblot analysis of the translation extracts using antibodies specific for PABP is shown at the top: lane 1, untreated; lane 2, treated with GST-Paip2; lane 3, treated with GST. For each transcript, the value obtained in the GST-treated extract was divided by the value obtained in the PABP-depleted extract. The stimulation values represent the average ± standard error of three independent duplicate experiments. (B) Krebs-2 cell-free translation reaction mixtures preincubated for 30 min at room temperature with the indicated amounts of purified HIS-Apc5 were programmed with 50 ng of the indicated transcripts for a further 50-min incubation at 37°C. Firefly luciferase activity in the absence of Apc5 was set as 100%. The values represent the average ± standard error of three independent experiments. (C) Krebs-2 cell-free translation reaction mixtures preincubated for 30 min at room tem-perature with 1 μg of HIS-Apc5 and the indicated amounts of purified GST-RRM3 were programmed with 50 ng of the indicated transcripts for a further 50-min incubation at 37°C. Firefly luciferase activity in the absence of HIS-Apc5 and GST-RRM3 was set as 100%. The values represent the average ± standard error of three independent experiments.
Figure Legend Snippet: Effect of PABP and Apc5 on translation in vitro. (A) Krebs-2 cell-free translation reaction mixtures that had been pretreated with GST-Paip2 for PABP depletion or with GST as a control were programmed with 100 ng of the indicated transcripts at 37°C for 90 min followed by measurements of firefly luciferase activity. Immunoblot analysis of the translation extracts using antibodies specific for PABP is shown at the top: lane 1, untreated; lane 2, treated with GST-Paip2; lane 3, treated with GST. For each transcript, the value obtained in the GST-treated extract was divided by the value obtained in the PABP-depleted extract. The stimulation values represent the average ± standard error of three independent duplicate experiments. (B) Krebs-2 cell-free translation reaction mixtures preincubated for 30 min at room temperature with the indicated amounts of purified HIS-Apc5 were programmed with 50 ng of the indicated transcripts for a further 50-min incubation at 37°C. Firefly luciferase activity in the absence of Apc5 was set as 100%. The values represent the average ± standard error of three independent experiments. (C) Krebs-2 cell-free translation reaction mixtures preincubated for 30 min at room tem-perature with 1 μg of HIS-Apc5 and the indicated amounts of purified GST-RRM3 were programmed with 50 ng of the indicated transcripts for a further 50-min incubation at 37°C. Firefly luciferase activity in the absence of HIS-Apc5 and GST-RRM3 was set as 100%. The values represent the average ± standard error of three independent experiments.

Techniques Used: In Vitro, Luciferase, Activity Assay, Purification, Incubation, Transmission Electron Microscopy

7) Product Images from "In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1"

Article Title: In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06554-11

Effects of PN-1 on angiogenic responses of HUVECs. (A) Concentration-dependent effect of PN-1 on 10 ng/ml FGF- or VEGF-induced HUVEC proliferation ( n = 4 to 10). (B) Effect of PN-1 (20 μg/ml) on cell proliferation induced by increasing growth factor (GF) concentrations ( n = 4). Results are expressed as the percentage of inhibition of proliferation, calculated from the ratio of the proliferation induced by each growth factor concentration measured in the presence of recombinant PN-1 to that measured in the absence of PN-1. (C and D) HUVEC adhesion (C) and HUVEC spreading on vitronectin and fibronectin (D), in the absence or presence of 20 μg/ml PN-1 ( n = 3 to 4). (E) HUVEC migration in the absence or presence of 20 ng/ml VEGF. *, P
Figure Legend Snippet: Effects of PN-1 on angiogenic responses of HUVECs. (A) Concentration-dependent effect of PN-1 on 10 ng/ml FGF- or VEGF-induced HUVEC proliferation ( n = 4 to 10). (B) Effect of PN-1 (20 μg/ml) on cell proliferation induced by increasing growth factor (GF) concentrations ( n = 4). Results are expressed as the percentage of inhibition of proliferation, calculated from the ratio of the proliferation induced by each growth factor concentration measured in the presence of recombinant PN-1 to that measured in the absence of PN-1. (C and D) HUVEC adhesion (C) and HUVEC spreading on vitronectin and fibronectin (D), in the absence or presence of 20 μg/ml PN-1 ( n = 3 to 4). (E) HUVEC migration in the absence or presence of 20 ng/ml VEGF. *, P

Techniques Used: Concentration Assay, Inhibition, Recombinant, Migration

Effects of PN-1 on HUVEC responses to VEGF. (A) Capillary tube formation in Matrigel in the presence or absence of 50 ng/ml VEGF and 20 μg/ml PN-1. Results are expressed as the mean tube density relative to the control without VEGF, measured on 3 fields per well from 3 to 4 experiments. ***, P
Figure Legend Snippet: Effects of PN-1 on HUVEC responses to VEGF. (A) Capillary tube formation in Matrigel in the presence or absence of 50 ng/ml VEGF and 20 μg/ml PN-1. Results are expressed as the mean tube density relative to the control without VEGF, measured on 3 fields per well from 3 to 4 experiments. ***, P

Techniques Used:

Characterization of PN-1 variants. (Inset) SDS-PAGE Coomassie staining of PN-1 after purification. (A and B) Thrombin inhibition by WT and K7Q PN-1 and catalytic effect of heparin. Thrombin and chromogenic substrate concentrations were 100 pM and 300 μM, respectively. Progress curves are shown. (A) Curves obtained with 50 nM WT (○) and K7Q (□) PN-1, used to determine uncatalyzed rate constants; (B) catalytic effect of 5 nM on thrombin inhibition by 2 nM PN-1. Dashed superimposed lines correspond to WT PN-1, K7Q PN-1, and K7Q PN-1 plus heparin. ●, WT PN1 plus heparin. (C) Representative fluorescence spectra of three of the TNS-bound PN-1 variants in the absence (solid lines) or presence (dashed lines) of heparin, determined with 500 nM WT or K7Q PN-1 and 750 nM R346A. Spectra are difference spectra between TNS plus PN-1 with or without heparin and TNS alone.
Figure Legend Snippet: Characterization of PN-1 variants. (Inset) SDS-PAGE Coomassie staining of PN-1 after purification. (A and B) Thrombin inhibition by WT and K7Q PN-1 and catalytic effect of heparin. Thrombin and chromogenic substrate concentrations were 100 pM and 300 μM, respectively. Progress curves are shown. (A) Curves obtained with 50 nM WT (○) and K7Q (□) PN-1, used to determine uncatalyzed rate constants; (B) catalytic effect of 5 nM on thrombin inhibition by 2 nM PN-1. Dashed superimposed lines correspond to WT PN-1, K7Q PN-1, and K7Q PN-1 plus heparin. ●, WT PN1 plus heparin. (C) Representative fluorescence spectra of three of the TNS-bound PN-1 variants in the absence (solid lines) or presence (dashed lines) of heparin, determined with 500 nM WT or K7Q PN-1 and 750 nM R346A. Spectra are difference spectra between TNS plus PN-1 with or without heparin and TNS alone.

Techniques Used: SDS Page, Staining, Purification, Inhibition, Fluorescence

Impact of PN-1 deficiency on in vivo angiogenesis: vessel formation in Matrigel plugs. Control plugs were implanted in 9 to 10 wild-type mice (PN-1 +/+) or PN-1-deficient mice (PN-1 -/-), and plugs supplemented with 20 μg/ml recombinant PN-1 (rPN-1) were implanted in 3 PN-1-deficient mice (PN-1 −/− + rPN-1). (A) Representative fluorescence of the vessels following retroorbital injection of FITC-dextran. (B) Representative plugs from PN-1 +/+ and PN-1 −/− mice. (C) Representative microphotographs of sections of Matrigel plugs stained with hematoxylin-eosin. Magnification, ×200. Arrows indicate erythrocyte-containing neovessels. (D) Quantification of cell infiltration (4 fields/plug). ***, P
Figure Legend Snippet: Impact of PN-1 deficiency on in vivo angiogenesis: vessel formation in Matrigel plugs. Control plugs were implanted in 9 to 10 wild-type mice (PN-1 +/+) or PN-1-deficient mice (PN-1 -/-), and plugs supplemented with 20 μg/ml recombinant PN-1 (rPN-1) were implanted in 3 PN-1-deficient mice (PN-1 −/− + rPN-1). (A) Representative fluorescence of the vessels following retroorbital injection of FITC-dextran. (B) Representative plugs from PN-1 +/+ and PN-1 −/− mice. (C) Representative microphotographs of sections of Matrigel plugs stained with hematoxylin-eosin. Magnification, ×200. Arrows indicate erythrocyte-containing neovessels. (D) Quantification of cell infiltration (4 fields/plug). ***, P

Techniques Used: In Vivo, Mouse Assay, Recombinant, Fluorescence, Injection, Staining

Impact of PN-1 deficiency on ex vivo angiogenesis. Thoracic aortic rings from PN-1-deficient (-/-) and WT (+/+) mice were cultured in a collagen gel in the presence or absence of 50 ng/ml VEGF and 20 μg/ml recombinant WT PN-1. The microvascular networks sprouting from the rings were observed by phase-contrast microscopy and after isolectin B4 labeling (in red), and results were quantified by densitometric analysis. Representative micrographs and results are shown for 6 to 20 rings from 3 to 7 mice. ***, P
Figure Legend Snippet: Impact of PN-1 deficiency on ex vivo angiogenesis. Thoracic aortic rings from PN-1-deficient (-/-) and WT (+/+) mice were cultured in a collagen gel in the presence or absence of 50 ng/ml VEGF and 20 μg/ml recombinant WT PN-1. The microvascular networks sprouting from the rings were observed by phase-contrast microscopy and after isolectin B4 labeling (in red), and results were quantified by densitometric analysis. Representative micrographs and results are shown for 6 to 20 rings from 3 to 7 mice. ***, P

Techniques Used: Ex Vivo, Mouse Assay, Cell Culture, Recombinant, Microscopy, Labeling

PN-1 binding to HUVECs at 4°C. (A to C) Cells were incubated at 4°C with recombinant PN-1 variants (1 μg/ml) in the presence or not of heparin (Hep), chondroitin sulfate (CS), or dermatan sulfate (DS) (A and C) or with supernatant of resting or activated platelets (B). Whole-cell lysates were submitted to immunoblotting with a polyclonal anti-PN-1 antibody and an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody as a protein loading control. Results are shown with representative immunoblots from at least 3 independent experiments (A, B, and C). (D) Densitometric quantification, expressed as the mean intensity (PN-1/GAPDH ratio) relative to control (WT binding), determined for each immunoblot. NS, not significant; **, P
Figure Legend Snippet: PN-1 binding to HUVECs at 4°C. (A to C) Cells were incubated at 4°C with recombinant PN-1 variants (1 μg/ml) in the presence or not of heparin (Hep), chondroitin sulfate (CS), or dermatan sulfate (DS) (A and C) or with supernatant of resting or activated platelets (B). Whole-cell lysates were submitted to immunoblotting with a polyclonal anti-PN-1 antibody and an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody as a protein loading control. Results are shown with representative immunoblots from at least 3 independent experiments (A, B, and C). (D) Densitometric quantification, expressed as the mean intensity (PN-1/GAPDH ratio) relative to control (WT binding), determined for each immunoblot. NS, not significant; **, P

Techniques Used: Binding Assay, Incubation, Recombinant, Western Blot

Role of PN-1 functional sites on PN-1 HUVEC angiogenic responses. Effects of PN-1 variants (20 μg/ml [A and C] or 50 μg/ml [B]) on FGF- and VEGF-induced HUVEC proliferation ( n = 4 to 10) (A), VEGF-induced HUVEC migration ( n = 3) (B), and VEGF-induced HUVEC organization in Matrigel (C). The tube density was measured on 3 fields per well from 3 to 4 experiments and averaged relative to the control without PN-1 and VEGF. (D) Effects of WT PN-1 on VEGF 121-induced HUVEC proliferation. **, P
Figure Legend Snippet: Role of PN-1 functional sites on PN-1 HUVEC angiogenic responses. Effects of PN-1 variants (20 μg/ml [A and C] or 50 μg/ml [B]) on FGF- and VEGF-induced HUVEC proliferation ( n = 4 to 10) (A), VEGF-induced HUVEC migration ( n = 3) (B), and VEGF-induced HUVEC organization in Matrigel (C). The tube density was measured on 3 fields per well from 3 to 4 experiments and averaged relative to the control without PN-1 and VEGF. (D) Effects of WT PN-1 on VEGF 121-induced HUVEC proliferation. **, P

Techniques Used: Functional Assay, Migration

8) Product Images from "Activator Protein 2α Associates with Adenomatous Polyposis Coli/β-Catenin and Inhibits β-Catenin/T-cell Factor Transcriptional Activity in Colorectal Cancer Cells *"

Article Title: Activator Protein 2α Associates with Adenomatous Polyposis Coli/β-Catenin and Inhibits β-Catenin/T-cell Factor Transcriptional Activity in Colorectal Cancer Cells *

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M405025200

Truncated forms of AP-2 α  fail to inhibit TOPflash reporter activity A , schematic presentation of full-length and truncated forms of AP-2 α  transfected into HEK293 cells with TOPflash,  β -galactosidase, and S33Y  β -catenin ( B ) or no exogenous  β -catenin ( C ). Lysates were prepared and analyzed for luciferase and  β -galactosidase activities 48 h post-transfection. Luciferase activity was normalized to the corresponding  β -galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D.,  n  = 3.
Figure Legend Snippet: Truncated forms of AP-2 α fail to inhibit TOPflash reporter activity A , schematic presentation of full-length and truncated forms of AP-2 α transfected into HEK293 cells with TOPflash, β -galactosidase, and S33Y β -catenin ( B ) or no exogenous β -catenin ( C ). Lysates were prepared and analyzed for luciferase and β -galactosidase activities 48 h post-transfection. Luciferase activity was normalized to the corresponding β -galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3.

Techniques Used: Activity Assay, Transfection, Luciferase

AP-2 α  binds to the N-terminal repeats in APC A , scheme showing wild type (full-length) APC and truncated proteins (APC1-APC5) with one or two of the repeat domains. *, positions of nuclear export signals.  B ,  35 S-labeled APC fragments as well as  β -catenin were synthesized  in vitro  and incubated with Sepharose-bound GST-AP-2 α . Beads were washed, and bound  35 S-labeled proteins were eluted, followed by SDS-PAGE and autoradiography. The  lower panel  shows 50% of the total radioactivity in each reaction (50% input controls).
Figure Legend Snippet: AP-2 α binds to the N-terminal repeats in APC A , scheme showing wild type (full-length) APC and truncated proteins (APC1-APC5) with one or two of the repeat domains. *, positions of nuclear export signals. B , 35 S-labeled APC fragments as well as β -catenin were synthesized in vitro and incubated with Sepharose-bound GST-AP-2 α . Beads were washed, and bound 35 S-labeled proteins were eluted, followed by SDS-PAGE and autoradiography. The lower panel shows 50% of the total radioactivity in each reaction (50% input controls).

Techniques Used: Labeling, Synthesized, In Vitro, Incubation, SDS Page, Autoradiography, Radioactivity

TOPflash reporter activation by exogenous β -catenin is suppressed by AP-2 α in HEK293 cells Cells were transfected with TOPflash, a β -catenin/TCF/LEF-responsive luciferase reporter plasmid containing TCF-4-binding sites, or the corresponding negative control FOPflash, as well as with AP-2 α and β -catenin constructs (as indicated in the figure). pSV- β -Gal was used as internal control. Luciferase and β -galactosidase activities were determined for whole cell lysates 48 h post-transfection, as described under “Experimental Procedures.” Luciferase activity was normalized to the corresponding β -galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3. A , induction of TOPflash activity by wild type β -catenin is suppressed by AP-2 α . The cell lysates also were immunoblotted for AP-2 α , β -catenin, and β -actin ( lower panel ). B , overexpression of AP-2 α inhibits TOPflash activity induced by oncogenic S33Y β -catenin. C , AP-2 α inhibits induction of TOPflash activity by Δ45 β -catenin.
Figure Legend Snippet: TOPflash reporter activation by exogenous β -catenin is suppressed by AP-2 α in HEK293 cells Cells were transfected with TOPflash, a β -catenin/TCF/LEF-responsive luciferase reporter plasmid containing TCF-4-binding sites, or the corresponding negative control FOPflash, as well as with AP-2 α and β -catenin constructs (as indicated in the figure). pSV- β -Gal was used as internal control. Luciferase and β -galactosidase activities were determined for whole cell lysates 48 h post-transfection, as described under “Experimental Procedures.” Luciferase activity was normalized to the corresponding β -galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3. A , induction of TOPflash activity by wild type β -catenin is suppressed by AP-2 α . The cell lysates also were immunoblotted for AP-2 α , β -catenin, and β -actin ( lower panel ). B , overexpression of AP-2 α inhibits TOPflash activity induced by oncogenic S33Y β -catenin. C , AP-2 α inhibits induction of TOPflash activity by Δ45 β -catenin.

Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Binding Assay, Negative Control, Construct, Activity Assay, Over Expression

Nuclear β -catenin and TCF-4 levels are unchanged by AP-2 α Cells were transfected with plasmid pcDNA3.1/AP-2 α or empty vector pcDNA3.1(+). After 48 h, nuclear lysates were prepared and immunoblotted for β -catenin, TCF-4, and AP-2 α , using β -actin as loading control.
Figure Legend Snippet: Nuclear β -catenin and TCF-4 levels are unchanged by AP-2 α Cells were transfected with plasmid pcDNA3.1/AP-2 α or empty vector pcDNA3.1(+). After 48 h, nuclear lysates were prepared and immunoblotted for β -catenin, TCF-4, and AP-2 α , using β -actin as loading control.

Techniques Used: Transfection, Plasmid Preparation

AP-2 α associates with APC and β -catenin, but not with TCF-4 Cell lysates from nontransfected cells were normalized for protein content and subjected to immunoprecipitation ( IP ) followed by immunoblotting ( IB ). A , immunoprecipitation was performed with the antibodies indicated, followed by immunoblotting with anti-TCF-4 antibody. Negative controls included no antibody ( No Ab ) and irrelevant antibody ( HA-Tag ). Note that AP-2 α antibody failed to pull down TCF-4. B , immunoprecipitation was first performed with the antibodies indicated, followed by immunoblotting with anti- β -catenin. β -Catenin was associated both with APC and with AP-2 α. C , immunoprecipitation with the antibodies indicated followed by immunoblotting with anti-APC identified full-length APC bound to AP-2 α in HEK293 and HCT116 cells. D , in HT29 cells, immunoprecipitation with anti-AP-2 α antibody pulled down 200- and 100-kDa truncated forms of APC, and anti-APC antibody pulled down AP-2 α (48 kDa).
Figure Legend Snippet: AP-2 α associates with APC and β -catenin, but not with TCF-4 Cell lysates from nontransfected cells were normalized for protein content and subjected to immunoprecipitation ( IP ) followed by immunoblotting ( IB ). A , immunoprecipitation was performed with the antibodies indicated, followed by immunoblotting with anti-TCF-4 antibody. Negative controls included no antibody ( No Ab ) and irrelevant antibody ( HA-Tag ). Note that AP-2 α antibody failed to pull down TCF-4. B , immunoprecipitation was first performed with the antibodies indicated, followed by immunoblotting with anti- β -catenin. β -Catenin was associated both with APC and with AP-2 α. C , immunoprecipitation with the antibodies indicated followed by immunoblotting with anti-APC identified full-length APC bound to AP-2 α in HEK293 and HCT116 cells. D , in HT29 cells, immunoprecipitation with anti-AP-2 α antibody pulled down 200- and 100-kDa truncated forms of APC, and anti-APC antibody pulled down AP-2 α (48 kDa).

Techniques Used: Immunoprecipitation

AP-2 α  and APC, alone or in combination, inhibits the interaction of  β -catenin with TCF-4 in the nucleus AP-2 α  and/or APC were overexpressed by transient transfection, as indicated, and anti-TCF-4 antibody was used to pull-down  β -catenin from nuclear extracts, 48 h post-transfection.  β -Catenin levels were analyzed by immunoblotting, and the same blots were reprobed for TCF-4. Nuclear lysates from control groups were used for negative controls of co-immu-noprecipitation, including no antibody ( No Ab ) and irrelevant antibody ( HA-Tag ). Relative  β -catenin levels were determined by densitometric analysis and normalized to TCF-4;  β -catenin expression recovered in the nontransfected controls was assigned an arbitrary value of 1.00.
Figure Legend Snippet: AP-2 α and APC, alone or in combination, inhibits the interaction of β -catenin with TCF-4 in the nucleus AP-2 α and/or APC were overexpressed by transient transfection, as indicated, and anti-TCF-4 antibody was used to pull-down β -catenin from nuclear extracts, 48 h post-transfection. β -Catenin levels were analyzed by immunoblotting, and the same blots were reprobed for TCF-4. Nuclear lysates from control groups were used for negative controls of co-immu-noprecipitation, including no antibody ( No Ab ) and irrelevant antibody ( HA-Tag ). Relative β -catenin levels were determined by densitometric analysis and normalized to TCF-4; β -catenin expression recovered in the nontransfected controls was assigned an arbitrary value of 1.00.

Techniques Used: Transfection, Expressing

9) Product Images from "The adaptor protein p40phox as a positive regulator of the superoxide-producing phagocyte oxidase"

Article Title: The adaptor protein p40phox as a positive regulator of the superoxide-producing phagocyte oxidase

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf642

Fig. 8 . p40 phox -enhanced superoxide production upon cell stimulation with the muscarinic receptor peptide m4I3C(14). ( A ) Superoxide production by the K562 cells with stable expression of p40 phox or without p40 phox . The K562 cells (5.0 × 10 3 cells) were stimulated with the muscarinic receptor peptide m4I3C(14) (m) (200 µM) and the chemiluminescence change was continuously monitored with DIOGENES, and SOD (50 µg/ml) was added where indicated (arrowhead). For details see Materials and methods. ( B ) Effect of pertussis toxin (PTX) on superoxide production upon stimulation with m4I3C(14) or PMA. The K562 cells (5.0 × 10 3 cells) were pretreated with PTX (18 µg/ml) and stimulated with m4I3C(14) (200 µM) or PMA (200 ng/ml) and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD. ( C ) Effect of the D289A substitution in the PC motif of p40 phox on superoxide production upon stimulation with m4I3C(14). The K562 cells with p40 phox (WT) or p40 phox (D289A) or without p40 phox (5.0 × 10 3 cells) were stimulated with 200 µM m4I3C(14) and superoxide production was measured as change of chemiluminescence. Each histogram represents the average from five independent experiments, with bars representing SD. ( D ) Effect of the K355A substitution in the PB1 domain of p67 phox on superoxide production upon stimulation with m4I3C(14). The K562 cells with p67 phox (WT) or p67 phox (K355A) or without p67 phox (5.0 × 10 3 cells) were stimulated with 200 µM m4I3C(14), and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD.
Figure Legend Snippet: Fig. 8 . p40 phox -enhanced superoxide production upon cell stimulation with the muscarinic receptor peptide m4I3C(14). ( A ) Superoxide production by the K562 cells with stable expression of p40 phox or without p40 phox . The K562 cells (5.0 × 10 3 cells) were stimulated with the muscarinic receptor peptide m4I3C(14) (m) (200 µM) and the chemiluminescence change was continuously monitored with DIOGENES, and SOD (50 µg/ml) was added where indicated (arrowhead). For details see Materials and methods. ( B ) Effect of pertussis toxin (PTX) on superoxide production upon stimulation with m4I3C(14) or PMA. The K562 cells (5.0 × 10 3 cells) were pretreated with PTX (18 µg/ml) and stimulated with m4I3C(14) (200 µM) or PMA (200 ng/ml) and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD. ( C ) Effect of the D289A substitution in the PC motif of p40 phox on superoxide production upon stimulation with m4I3C(14). The K562 cells with p40 phox (WT) or p40 phox (D289A) or without p40 phox (5.0 × 10 3 cells) were stimulated with 200 µM m4I3C(14) and superoxide production was measured as change of chemiluminescence. Each histogram represents the average from five independent experiments, with bars representing SD. ( D ) Effect of the K355A substitution in the PB1 domain of p67 phox on superoxide production upon stimulation with m4I3C(14). The K562 cells with p67 phox (WT) or p67 phox (K355A) or without p67 phox (5.0 × 10 3 cells) were stimulated with 200 µM m4I3C(14), and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD.

Techniques Used: Cell Stimulation, Expressing

Fig. 6 . Effect of the K355A substitution in the PB1 domain of p67 phox on its interaction with p40 phox . ( A ) Expression of p67 phox in K562 cells. K562 cells stably expressing gp91 phox , p47 phox and p40 phox were transfected with pREP4 encoding p67 phox (WT) or p67 phox (K355A). The K562 cells were stained with anti-p67 phox serum (filled histogram) or pre-immune serum (open histogram), and analyzed by flow cytometry. ( B ) In vivo interaction of p67 phox with p40 phox in the K562 cells. The cell lysates of the K562 cells were analyzed by immunoprecipitation with the anti-p67 phox or control IgG (cont.) (left panel), or the anti-p47 phox antibody or control IgG (cont.) (right panel) followed by immunoblot (Blot) with the indicated antibody.
Figure Legend Snippet: Fig. 6 . Effect of the K355A substitution in the PB1 domain of p67 phox on its interaction with p40 phox . ( A ) Expression of p67 phox in K562 cells. K562 cells stably expressing gp91 phox , p47 phox and p40 phox were transfected with pREP4 encoding p67 phox (WT) or p67 phox (K355A). The K562 cells were stained with anti-p67 phox serum (filled histogram) or pre-immune serum (open histogram), and analyzed by flow cytometry. ( B ) In vivo interaction of p67 phox with p40 phox in the K562 cells. The cell lysates of the K562 cells were analyzed by immunoprecipitation with the anti-p67 phox or control IgG (cont.) (left panel), or the anti-p47 phox antibody or control IgG (cont.) (right panel) followed by immunoblot (Blot) with the indicated antibody.

Techniques Used: Expressing, Stable Transfection, Transfection, Staining, Flow Cytometry, Cytometry, In Vivo, Immunoprecipitation

Fig. 7 . Effect of the K355A substitution in the PB1 domain on membrane translocation of p47 phox and p67 phox and superoxide production upon PMA stimulation. ( A ) Membrane translocation of p47 phox , p67 phox and p40 phox upon PMA stimulation. The K562 cells stably expressing p67 phox (WT) or p67 phox (K355A) (1.0 × 10 7 cells) were stimulated with PMA (200 ng/ml) for the indicated time and the amounts of p47 phox , p67 phox and p40 phox in the membrane fractions were analyzed by immunoblot. ( B ) Superoxide production by the K562 cells with p67 phox (WT) or p67 phox (K355A) was measured as change of chemiluminescence. The K562 cells (5.0 × 10 3 cells) were stimulated with PMA (200 ng/ml) and the chemiluminescence change was measured. The chemiluminescence change by K562 cells without the expression of p40 phox and with p67 phox (WT) or p67 phox (K355A) was also measured. Each histogram indicates the average from five independent experiments, with bars representing SD.
Figure Legend Snippet: Fig. 7 . Effect of the K355A substitution in the PB1 domain on membrane translocation of p47 phox and p67 phox and superoxide production upon PMA stimulation. ( A ) Membrane translocation of p47 phox , p67 phox and p40 phox upon PMA stimulation. The K562 cells stably expressing p67 phox (WT) or p67 phox (K355A) (1.0 × 10 7 cells) were stimulated with PMA (200 ng/ml) for the indicated time and the amounts of p47 phox , p67 phox and p40 phox in the membrane fractions were analyzed by immunoblot. ( B ) Superoxide production by the K562 cells with p67 phox (WT) or p67 phox (K355A) was measured as change of chemiluminescence. The K562 cells (5.0 × 10 3 cells) were stimulated with PMA (200 ng/ml) and the chemiluminescence change was measured. The chemiluminescence change by K562 cells without the expression of p40 phox and with p67 phox (WT) or p67 phox (K355A) was also measured. Each histogram indicates the average from five independent experiments, with bars representing SD.

Techniques Used: Translocation Assay, Stable Transfection, Expressing

10) Product Images from "Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium"

Article Title: Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium

Journal: Genes & Development

doi: 10.1101/gad.871101

Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing GST–FbxA:F-box/WD40 were lysed and the 10,000 × G supernatant was adsorbed to g–Sepharose. The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.
Figure Legend Snippet: Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing GST–FbxA:F-box/WD40 were lysed and the 10,000 × G supernatant was adsorbed to g–Sepharose. The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.

Techniques Used: Western Blot, Mutagenesis, Expressing, Transformation Assay

11) Product Images from "Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation"

Article Title: Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks484

SUMO-PCNA inhibits Srs2 sumoylation by binding to the SIM of Srs2. ( A ) SUMO-PCNA inhibits Srs2 sumoylation in vitro . In vitro sumoylation assay was performed using Aos1/Uba2 (0.35 μM), Ubc9 (1.25 μM), SUMO (1.6 μM), Siz1 (1–465) (0.4 μM), Srs2 (0.75 μM) and ATP (100 μM) in the absence or presence of increasing amounts of SUMO-PCNA (0.8, 1.6, 3.2 μM, lanes 2–4) or PCNA-K164R (lanes 8–10). The reactions were stopped, resolved on 10% SDS-PAGE gel and stained with Coomassie Blue. ( B ) SUMO-PCNA outcompetes SUMO in Srs2 binding. Pull-down experiments using purified GST-SUMO (2 μM, lanes 1–4) and Srs2 (0.6 μM) in the absence (lanes 1 and 2) or presence of SUMO-PCNA (0.6 μM, lanes 3–6) were performed as in Figure 3 . ( C ) Lack of PCNA sumoylation alters the Srs2 sumoylation profile in vivo . Endogenous Srs2 from wild-type or pol30-K127, 164R yeast strains treated with 0.3% MMS was analyzed as in Figure 2 B. ( D ) The inhibitory effect of SUMO-PCNA on Srs2 sumoylation can be overcome by increasing amounts of SUMO and Siz1. Srs2 (0.75 μM) was pre-incubated with SUMO-PCNA (1.5 μM, lanes 1–4) for 15 min at RT, after which the sumoylation assay was performed using Aos1/Uba2 (0.35 μM), indicated amounts of Ubc9, SUMO and Siz1(1–465) in buffer containing 100 μM ATP and 300 mM KCl. Reactions were stopped and analyzed by 10% SDS-PAGE, followed by Coomassie Blue staining.
Figure Legend Snippet: SUMO-PCNA inhibits Srs2 sumoylation by binding to the SIM of Srs2. ( A ) SUMO-PCNA inhibits Srs2 sumoylation in vitro . In vitro sumoylation assay was performed using Aos1/Uba2 (0.35 μM), Ubc9 (1.25 μM), SUMO (1.6 μM), Siz1 (1–465) (0.4 μM), Srs2 (0.75 μM) and ATP (100 μM) in the absence or presence of increasing amounts of SUMO-PCNA (0.8, 1.6, 3.2 μM, lanes 2–4) or PCNA-K164R (lanes 8–10). The reactions were stopped, resolved on 10% SDS-PAGE gel and stained with Coomassie Blue. ( B ) SUMO-PCNA outcompetes SUMO in Srs2 binding. Pull-down experiments using purified GST-SUMO (2 μM, lanes 1–4) and Srs2 (0.6 μM) in the absence (lanes 1 and 2) or presence of SUMO-PCNA (0.6 μM, lanes 3–6) were performed as in Figure 3 . ( C ) Lack of PCNA sumoylation alters the Srs2 sumoylation profile in vivo . Endogenous Srs2 from wild-type or pol30-K127, 164R yeast strains treated with 0.3% MMS was analyzed as in Figure 2 B. ( D ) The inhibitory effect of SUMO-PCNA on Srs2 sumoylation can be overcome by increasing amounts of SUMO and Siz1. Srs2 (0.75 μM) was pre-incubated with SUMO-PCNA (1.5 μM, lanes 1–4) for 15 min at RT, after which the sumoylation assay was performed using Aos1/Uba2 (0.35 μM), indicated amounts of Ubc9, SUMO and Siz1(1–465) in buffer containing 100 μM ATP and 300 mM KCl. Reactions were stopped and analyzed by 10% SDS-PAGE, followed by Coomassie Blue staining.

Techniques Used: Binding Assay, In Vitro, SDS Page, Staining, Purification, In Vivo, Incubation

The interaction between the Srs2 SIM motif and SUMO is necessary for Srs2 sumoylation. ( A ) SUMO interacts with the Srs2 SIM motif. Purified GST-SUMO (2 μM, lanes 1, 2 and 5, 6) was incubated with Srs2 (0.6 μM, lanes 1–4) or its mutant lacking the SIM motif—Srs2ΔSIM (0.6 μM, lanes 5–8) and GTH-Sepharose in buffer S2 containing 100 mM KCl for 30 min at RT. The beads were washed and treated with SDS Laemmli buffer to elute the bound proteins. The supernatant (S) containing unbound Srs2 protein, and the SDS eluate (E) (10 μl each) were analyzed by 10% SDS–PAGE followed by staining with Coomassie Blue. Reactions containing only GTH-Sepharose and Srs2 (lanes 3 and 4) or Srs2ΔSIM (lanes 7 and 8) were included as negative control. Numbers on the left side of the gel indicate molecular weights (in kDa) of protein standards. ( B ) Ubc9 does not interact with Srs2. Interaction between purified GST-Ubc9 (4 μM, lanes 1–3) and Srs2 (1.25 μM) was analyzed as in (A). ( C ) SUMO modification of Ubc9 triggers its interaction with Srs2. GST-Ubc9 (4 μM, lanes 1–2) or GST-Ubc9-SUMO (4 μM, lanes 3–6), prepared by sumoylation reaction in the absence or presence of ATP, was mixed with Srs2 (1.25 μM, lanes 1–4) or Srs2ΔSIM (1.25 μM, lanes 5–6) and analyzed as in (A), except β-mercaptoethanol was excluded from the Laemmli buffer to prevent denaturation of SUMO-charged Ubc9. ( D ) Yeast two-hybrid interaction of Ubc9 and SUMO with Srs2 is mediated by its SIM motif. Strain PJ69-4 containing UBC9 or SUMO fused to the GAL4 transcription activation domain and SRS2 (aa 783–1174) or SRS2ΔSIM (a.a. 783–1169) fused to the GAL4 DNA-binding domain, were spotted as 10-fold serial dilutions on medium lacking leucine and tryptophan or leucine, tryptophan and histidine. The empty vector (pGADT7) was included as negative control. ( E ) Srs2 SIM motif is necessary for Srs2 sumoylation in vitro . The standard in vitro sumoylation reaction was done with wild-type Srs2 (lanes 1 and 2) or Srs2ΔSIM (lanes 3 and 4) in buffer S2 containing 100 mM KCl. ( F ) In vivo sumoylation of Srs2 requires its SIM motif. Yeast cells, expressing His-tagged wild-type Srs2 or Srs2ΔSIM mutant under the copper-responsive CUP1 promoter, were grown in the absence or presence of 0.3% MMS and immunoprecipitated using anti-Srs2 antibody. Western blotting was performed as in Figure 2 B.
Figure Legend Snippet: The interaction between the Srs2 SIM motif and SUMO is necessary for Srs2 sumoylation. ( A ) SUMO interacts with the Srs2 SIM motif. Purified GST-SUMO (2 μM, lanes 1, 2 and 5, 6) was incubated with Srs2 (0.6 μM, lanes 1–4) or its mutant lacking the SIM motif—Srs2ΔSIM (0.6 μM, lanes 5–8) and GTH-Sepharose in buffer S2 containing 100 mM KCl for 30 min at RT. The beads were washed and treated with SDS Laemmli buffer to elute the bound proteins. The supernatant (S) containing unbound Srs2 protein, and the SDS eluate (E) (10 μl each) were analyzed by 10% SDS–PAGE followed by staining with Coomassie Blue. Reactions containing only GTH-Sepharose and Srs2 (lanes 3 and 4) or Srs2ΔSIM (lanes 7 and 8) were included as negative control. Numbers on the left side of the gel indicate molecular weights (in kDa) of protein standards. ( B ) Ubc9 does not interact with Srs2. Interaction between purified GST-Ubc9 (4 μM, lanes 1–3) and Srs2 (1.25 μM) was analyzed as in (A). ( C ) SUMO modification of Ubc9 triggers its interaction with Srs2. GST-Ubc9 (4 μM, lanes 1–2) or GST-Ubc9-SUMO (4 μM, lanes 3–6), prepared by sumoylation reaction in the absence or presence of ATP, was mixed with Srs2 (1.25 μM, lanes 1–4) or Srs2ΔSIM (1.25 μM, lanes 5–6) and analyzed as in (A), except β-mercaptoethanol was excluded from the Laemmli buffer to prevent denaturation of SUMO-charged Ubc9. ( D ) Yeast two-hybrid interaction of Ubc9 and SUMO with Srs2 is mediated by its SIM motif. Strain PJ69-4 containing UBC9 or SUMO fused to the GAL4 transcription activation domain and SRS2 (aa 783–1174) or SRS2ΔSIM (a.a. 783–1169) fused to the GAL4 DNA-binding domain, were spotted as 10-fold serial dilutions on medium lacking leucine and tryptophan or leucine, tryptophan and histidine. The empty vector (pGADT7) was included as negative control. ( E ) Srs2 SIM motif is necessary for Srs2 sumoylation in vitro . The standard in vitro sumoylation reaction was done with wild-type Srs2 (lanes 1 and 2) or Srs2ΔSIM (lanes 3 and 4) in buffer S2 containing 100 mM KCl. ( F ) In vivo sumoylation of Srs2 requires its SIM motif. Yeast cells, expressing His-tagged wild-type Srs2 or Srs2ΔSIM mutant under the copper-responsive CUP1 promoter, were grown in the absence or presence of 0.3% MMS and immunoprecipitated using anti-Srs2 antibody. Western blotting was performed as in Figure 2 B.

Techniques Used: Purification, Incubation, Mutagenesis, SDS Page, Staining, Negative Control, Modification, Activation Assay, Binding Assay, Plasmid Preparation, In Vitro, In Vivo, Expressing, Immunoprecipitation, Western Blot

Sumoylation of Srs2 inhibits its interaction with SUMO-PCNA and SUMO in vitro . ( A ) Sumoylation of Srs2 inhibits its interaction with SUMO-PCNA. His-tagged Srs2 (1.5 μM, lanes 1 and 2) or SUMO-Srs2 (1.5 μM, lanes 3 and 4), prepared by sumoylation reaction using untagged SUMO and Siz1 proteins, was mixed with SUMO-PCNA (1.5 μM) and Ni-charged resin. The beads were washed and treated with SDS Laemmli buffer to elute the bound proteins. The supernatant (S) containing unbound Srs2 protein and the SDS eluate (E) (10 μl each) were analyzed by 10% SDS–PAGE followed by Coomassie Blue staining. Sumoylation reaction in the absence of Srs2 was mixed with Ni-charged resin as a negative control (lanes 5 and 6). ( B ) Sumoylation of Srs2 inhibits its interaction with SUMO. Srs2 (1.5 μM, lanes 1–4) or SUMO-Srs2 (1.5 μM, lanes 5–8), prepared by sumoylation reaction in the absence or presence of ATP, was mixed with GST-SUMO (1.5 μM, lanes 1, 2, 5 and 6) or without it (lanes 3, 4, 7 and 8) and pulled-down on GTH-Sepharose beads. The analysis was performed as in (A).
Figure Legend Snippet: Sumoylation of Srs2 inhibits its interaction with SUMO-PCNA and SUMO in vitro . ( A ) Sumoylation of Srs2 inhibits its interaction with SUMO-PCNA. His-tagged Srs2 (1.5 μM, lanes 1 and 2) or SUMO-Srs2 (1.5 μM, lanes 3 and 4), prepared by sumoylation reaction using untagged SUMO and Siz1 proteins, was mixed with SUMO-PCNA (1.5 μM) and Ni-charged resin. The beads were washed and treated with SDS Laemmli buffer to elute the bound proteins. The supernatant (S) containing unbound Srs2 protein and the SDS eluate (E) (10 μl each) were analyzed by 10% SDS–PAGE followed by Coomassie Blue staining. Sumoylation reaction in the absence of Srs2 was mixed with Ni-charged resin as a negative control (lanes 5 and 6). ( B ) Sumoylation of Srs2 inhibits its interaction with SUMO. Srs2 (1.5 μM, lanes 1–4) or SUMO-Srs2 (1.5 μM, lanes 5–8), prepared by sumoylation reaction in the absence or presence of ATP, was mixed with GST-SUMO (1.5 μM, lanes 1, 2, 5 and 6) or without it (lanes 3, 4, 7 and 8) and pulled-down on GTH-Sepharose beads. The analysis was performed as in (A).

Techniques Used: In Vitro, SDS Page, Staining, Negative Control

12) 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

13) Product Images from "Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis"

Article Title: Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

Journal: Nature cell biology

doi: 10.1038/ncb2568

In vivo and In vitro function of AtWNK8 ( A ) In vivo phosphorylation of AtRGS1. Seedlings expressing AtRGS1-TAP were pretreated with 100 nM calyculin A and 10 mM sodium orthovanadate for 3 h followed by 6% D-glucose stimulation for 90 min. AtRGS1-TAP or AtGPA1 in seedling lysates was separated on a 12.5% Anderson’s gel and detected by immunoblot with peroxidase anti-peroxidase or anti-AtGPA1 antibody. ( B ) Four-day-old AtRGS1-YFP expressing seedlings were treated with phosphatase inhibitors, calyculin A, for 2 h followed by 6% glucose treatment or not (No glucose) for 1 h prior to imaging epidermal cells. Scale bars = 10 µm. Error = SEM, n = 5. ( C ) Phylogenetic tree of the AtWNK-family kinases. Full-length amino acid sequences were aligned with CLUSTAL W implemented in CLC Genomics Workbench using the following settings; Gap open penalty, 10; Gap extension penalty 1. The neighbor joining tree (1000 bootstrap replicate) was created with the aligned sequences. ( D ) In vitro binding between AtRGS1 and AtWNKs. Recombinant RGSbox+Cterm was tested for interaction with GST (negative control) or GST-AtWNKs using glutathione-Sepharose, and detected by immunoblot analysis using an anti-AtRGS1 antibody. ( E ) In vitro phosphorylation of AtRGS1 by AtWNK kinases. Recombinant GST or His-RGSbox+Cterm was incubated with GST-AtWNKs in reaction buffer containing γ 32 P-ATP. Proteins were separated on SDS-PAGE. ( F ) Radioactivity incorporated into the GST/RGS1 bands. Phosphorylation levels of three independent experiments were quantified in (E). Error bars = SEM. ( G ) Quantitation of sugar-induced AtRGS1 internalization in AtWNK-null mutants. Seedlings of Col-0, wnk1-1 , wnk8-1 , wnk8-2 or wnk10-2 transiently expressing AtRGS1-YFP were treated with 6% D-glucose for 30 min. WNK# denotes AtWNK members in panels C-F. Error bars = SEM, n = 5. Quantitation of fluorescence is described in Methods .
Figure Legend Snippet: In vivo and In vitro function of AtWNK8 ( A ) In vivo phosphorylation of AtRGS1. Seedlings expressing AtRGS1-TAP were pretreated with 100 nM calyculin A and 10 mM sodium orthovanadate for 3 h followed by 6% D-glucose stimulation for 90 min. AtRGS1-TAP or AtGPA1 in seedling lysates was separated on a 12.5% Anderson’s gel and detected by immunoblot with peroxidase anti-peroxidase or anti-AtGPA1 antibody. ( B ) Four-day-old AtRGS1-YFP expressing seedlings were treated with phosphatase inhibitors, calyculin A, for 2 h followed by 6% glucose treatment or not (No glucose) for 1 h prior to imaging epidermal cells. Scale bars = 10 µm. Error = SEM, n = 5. ( C ) Phylogenetic tree of the AtWNK-family kinases. Full-length amino acid sequences were aligned with CLUSTAL W implemented in CLC Genomics Workbench using the following settings; Gap open penalty, 10; Gap extension penalty 1. The neighbor joining tree (1000 bootstrap replicate) was created with the aligned sequences. ( D ) In vitro binding between AtRGS1 and AtWNKs. Recombinant RGSbox+Cterm was tested for interaction with GST (negative control) or GST-AtWNKs using glutathione-Sepharose, and detected by immunoblot analysis using an anti-AtRGS1 antibody. ( E ) In vitro phosphorylation of AtRGS1 by AtWNK kinases. Recombinant GST or His-RGSbox+Cterm was incubated with GST-AtWNKs in reaction buffer containing γ 32 P-ATP. Proteins were separated on SDS-PAGE. ( F ) Radioactivity incorporated into the GST/RGS1 bands. Phosphorylation levels of three independent experiments were quantified in (E). Error bars = SEM. ( G ) Quantitation of sugar-induced AtRGS1 internalization in AtWNK-null mutants. Seedlings of Col-0, wnk1-1 , wnk8-1 , wnk8-2 or wnk10-2 transiently expressing AtRGS1-YFP were treated with 6% D-glucose for 30 min. WNK# denotes AtWNK members in panels C-F. Error bars = SEM, n = 5. Quantitation of fluorescence is described in Methods .

Techniques Used: In Vivo, In Vitro, Expressing, Imaging, Binding Assay, Recombinant, Negative Control, Incubation, SDS Page, Radioactivity, Quantitation Assay, Fluorescence

14) Product Images from "U1 small nuclear RNP from Trypanosoma brucei: a minimal U1 snRNA with unusual protein components"

Article Title: U1 small nuclear RNP from Trypanosoma brucei: a minimal U1 snRNA with unusual protein components

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki548

Identification and U1 snRNP association of a novel, U1-specific protein component: TbU1-24K. ( A ) ClustalW alignment of the novel protein component of the T.brucei U1 snRNP, TbU1-24K, with two putative homologs from T.cruzi . Two conserved regions are indicated by the boxed regions; the positions with asterisks are identical. Accession numbers (Gene DB): T.cruzi 877 (Tc00.1047053503877.10), T.cruzi 455 (Tc00.1047053509455.110) and T.brucei (Tb03.27F10.160). ( B ) TbU1-24K is a U1 snRNP-specific component. Extract was prepared from a T.brucei cell line, which stably expresses TAP-tagged TbU1-24K protein, and used to affinity-purify TAP-tagged complexes. Copurifying RNAs were analyzed by northern blotting, using a mixed probe (snRNA positions indicated on the right). Lane 1, 1% of input; lane 2, all of IgG-selected and TEV-released material; M , DIG marker V (Roche). ( C ) TbU1-24K coexists with TbU1-70K and Sm proteins in the same RNP complex. TAP-tag affinity purification of TbU1-24K complexes and RNA analysis was carried out as described in (A) (lane 1, 1% input; lane 2, 10% of IgG-selected and TEV-released material). Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. The snRNA positions are marked on the right. The slightly slower mobility of U1 snRNA in the immunoprecipitates (lanes 4 and 5) is most likely caused by comigrating tRNA released from the blocked protein A–Sepharose beads. M , DIG marker V (Roche). ( D ) In vitro U1 snRNA binding of U1 snRNP proteins. GST derivatives of the three U1-specific proteins TbU1-24K, TbU1C and TbU1-70K (lanes 3–5) were incubated with in vitro transcribed T.brucei U1 snRNA, followed by GST pull-down and analysis of coprecipitated RNA by northern hybridization with a U1-specific probe. A total of 10% of the input material was analyzed (lane 1), and a control precipitation was carried out with GST protein (lane 2). M , DIG marker V (Roche).
Figure Legend Snippet: Identification and U1 snRNP association of a novel, U1-specific protein component: TbU1-24K. ( A ) ClustalW alignment of the novel protein component of the T.brucei U1 snRNP, TbU1-24K, with two putative homologs from T.cruzi . Two conserved regions are indicated by the boxed regions; the positions with asterisks are identical. Accession numbers (Gene DB): T.cruzi 877 (Tc00.1047053503877.10), T.cruzi 455 (Tc00.1047053509455.110) and T.brucei (Tb03.27F10.160). ( B ) TbU1-24K is a U1 snRNP-specific component. Extract was prepared from a T.brucei cell line, which stably expresses TAP-tagged TbU1-24K protein, and used to affinity-purify TAP-tagged complexes. Copurifying RNAs were analyzed by northern blotting, using a mixed probe (snRNA positions indicated on the right). Lane 1, 1% of input; lane 2, all of IgG-selected and TEV-released material; M , DIG marker V (Roche). ( C ) TbU1-24K coexists with TbU1-70K and Sm proteins in the same RNP complex. TAP-tag affinity purification of TbU1-24K complexes and RNA analysis was carried out as described in (A) (lane 1, 1% input; lane 2, 10% of IgG-selected and TEV-released material). Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. The snRNA positions are marked on the right. The slightly slower mobility of U1 snRNA in the immunoprecipitates (lanes 4 and 5) is most likely caused by comigrating tRNA released from the blocked protein A–Sepharose beads. M , DIG marker V (Roche). ( D ) In vitro U1 snRNA binding of U1 snRNP proteins. GST derivatives of the three U1-specific proteins TbU1-24K, TbU1C and TbU1-70K (lanes 3–5) were incubated with in vitro transcribed T.brucei U1 snRNA, followed by GST pull-down and analysis of coprecipitated RNA by northern hybridization with a U1-specific probe. A total of 10% of the input material was analyzed (lane 1), and a control precipitation was carried out with GST protein (lane 2). M , DIG marker V (Roche).

Techniques Used: Stable Transfection, Northern Blot, Marker, Affinity Purification, Immunoprecipitation, In Vitro, Binding Assay, Incubation, Hybridization

T.brucei U1C (TbU1C): a U1 snRNP-specific component binding specifically to the 5′ terminal sequence of U1 snRNA. ( A ) ClustalW alignment of the protein sequences for the newly identified U1C homologs from T.brucei , T.cruzi and L.major , in comparison with the human U1C sequence. The conserved C 2 H 2 -type Zn finger within the boxed sequence is highlighted by large-size letters; asterisks indicate absolutely conserved amino acid positions. Accession numbers (GeneDB): T.brucei (Tb10.70.5640), T.cruzi (Tc00.1047053511367.354) and L.major (LmjF21.0320); human U1C (P09234). ( B ) Extract was prepared from a T.brucei cell line, which stably expresses TAP-tagged TbU1C protein, and used to affinity-purify TAP-tagged complexes. Purification was followed by analyzing copurifying RNAs by northern blotting, using a mixed snRNA probe (snRNA positions indicated on the right). M , DIG marker V (Roche). Lane 1, 1% of input; lane 2, 10% of IgG-selected and TEV-released material. Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. ( C ) TbU1C protein binds specifically to the 5′ terminal sequence of U1 snRNA. GST TbU1C protein was incubated with 32 P-labeled full-length U1 snRNA (lanes 1 and 2) and various U1 snRNA derivatives: U1 Δstem–loop (lanes 3 and 4), U1 Δ5′(1–14) (lanes 5, 6), U1 Δ5′(1–30) (lanes 7 and 8), U1 5′ stem–loop (lanes 9 and 10), U1 5′(1–14) (lanes 11 and 12) or a 17mer control RNA (lanes 13 and 14). In each case, 10% of the input ( I ) and the total GST pull-down material ( P ) were analyzed.
Figure Legend Snippet: T.brucei U1C (TbU1C): a U1 snRNP-specific component binding specifically to the 5′ terminal sequence of U1 snRNA. ( A ) ClustalW alignment of the protein sequences for the newly identified U1C homologs from T.brucei , T.cruzi and L.major , in comparison with the human U1C sequence. The conserved C 2 H 2 -type Zn finger within the boxed sequence is highlighted by large-size letters; asterisks indicate absolutely conserved amino acid positions. Accession numbers (GeneDB): T.brucei (Tb10.70.5640), T.cruzi (Tc00.1047053511367.354) and L.major (LmjF21.0320); human U1C (P09234). ( B ) Extract was prepared from a T.brucei cell line, which stably expresses TAP-tagged TbU1C protein, and used to affinity-purify TAP-tagged complexes. Purification was followed by analyzing copurifying RNAs by northern blotting, using a mixed snRNA probe (snRNA positions indicated on the right). M , DIG marker V (Roche). Lane 1, 1% of input; lane 2, 10% of IgG-selected and TEV-released material. Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. ( C ) TbU1C protein binds specifically to the 5′ terminal sequence of U1 snRNA. GST TbU1C protein was incubated with 32 P-labeled full-length U1 snRNA (lanes 1 and 2) and various U1 snRNA derivatives: U1 Δstem–loop (lanes 3 and 4), U1 Δ5′(1–14) (lanes 5, 6), U1 Δ5′(1–30) (lanes 7 and 8), U1 5′ stem–loop (lanes 9 and 10), U1 5′(1–14) (lanes 11 and 12) or a 17mer control RNA (lanes 13 and 14). In each case, 10% of the input ( I ) and the total GST pull-down material ( P ) were analyzed.

Techniques Used: Binding Assay, Sequencing, Stable Transfection, Purification, Northern Blot, Marker, Affinity Purification, Immunoprecipitation, Incubation, Labeling

TbU1-70K is a U1 snRNP-specific protein and binds in vitro specifically to the 5′ loop sequence of U1 snRNA. ( A ) Comparison of the domain structures of T.brucei (Tb08.4A8.530) and the human U1-70K (A25707) proteins. ( B ) Western blot analysis of T.brucei U1 snRNP proteins. U1 snRNPs were affinity-purified from T.brucei extract by a 2′- O -methyl RNA antisense oligonucleotide, protein was prepared and analyzed by SDS–PAGE and western blotting, using polyclonal rabbit antibodies against TbU1-70K (U1-70K) or non-immune serum (NIS). The arrow points to the immunostained TbU1-70K band of apparent molecular weight 40 kDa. Protein markers are on the right (in kDa). ( C ) U1 snRNA is specifically coprecipitated from T.brucei extract by anti-Tb U1-70 antibodies. Immunoprecipitations were carried out from T.brucei extract, using NIS, or with antibodies against the TbU1-70K protein (U1-70K) or against the trypanosome Sm proteins (Sm). RNA was purified from the immunoprecipitates and analyzed by 3′ end labeling with [ 32 P]pCp. The positions of the SL RNA and snRNAs are marked on the right. M , 32 P-labeled pBR322/HpaII markers. ( D ) RNA from the same immunoprecipitates was also analyzed by primer extension with a U1-specific oligonucleotide. In addition, RNA from a 10% aliquot of the input was included; the positions of the primer ( p ) and the U1-specific primer-extension product (U1) are marked on the right. M , 32 P-labeled pBR322/HpaII markers. ( E ) 32 P-labeled T.brucei U1 snRNA and mutant derivatives [as indicated above the lanes; see (F)] were in vitro transcribed and incubated with GST-TbU1-70K, followed by GST pull-down. For each reaction, 10% of the input ( I ) and the total precipitated material ( P ) were analyzed. M , 32 P-labeled pBR322/HpaII markers. ( F ) Sequences and proposed secondary structures of the T.brucei U1 snRNA and its mutant derivatives. The boxed sequence in the T.brucei U1 snRNA indicates the Sm site; the two arrows indicate a potential second stem–loop. Below, the sequences of the stem–loop derivatives are given; the circled nucleotides mark the two positions in the human loop that differ from the T.brucei sequence, and the single-nucleotide mutation (A21) in the mutant human loop.
Figure Legend Snippet: TbU1-70K is a U1 snRNP-specific protein and binds in vitro specifically to the 5′ loop sequence of U1 snRNA. ( A ) Comparison of the domain structures of T.brucei (Tb08.4A8.530) and the human U1-70K (A25707) proteins. ( B ) Western blot analysis of T.brucei U1 snRNP proteins. U1 snRNPs were affinity-purified from T.brucei extract by a 2′- O -methyl RNA antisense oligonucleotide, protein was prepared and analyzed by SDS–PAGE and western blotting, using polyclonal rabbit antibodies against TbU1-70K (U1-70K) or non-immune serum (NIS). The arrow points to the immunostained TbU1-70K band of apparent molecular weight 40 kDa. Protein markers are on the right (in kDa). ( C ) U1 snRNA is specifically coprecipitated from T.brucei extract by anti-Tb U1-70 antibodies. Immunoprecipitations were carried out from T.brucei extract, using NIS, or with antibodies against the TbU1-70K protein (U1-70K) or against the trypanosome Sm proteins (Sm). RNA was purified from the immunoprecipitates and analyzed by 3′ end labeling with [ 32 P]pCp. The positions of the SL RNA and snRNAs are marked on the right. M , 32 P-labeled pBR322/HpaII markers. ( D ) RNA from the same immunoprecipitates was also analyzed by primer extension with a U1-specific oligonucleotide. In addition, RNA from a 10% aliquot of the input was included; the positions of the primer ( p ) and the U1-specific primer-extension product (U1) are marked on the right. M , 32 P-labeled pBR322/HpaII markers. ( E ) 32 P-labeled T.brucei U1 snRNA and mutant derivatives [as indicated above the lanes; see (F)] were in vitro transcribed and incubated with GST-TbU1-70K, followed by GST pull-down. For each reaction, 10% of the input ( I ) and the total precipitated material ( P ) were analyzed. M , 32 P-labeled pBR322/HpaII markers. ( F ) Sequences and proposed secondary structures of the T.brucei U1 snRNA and its mutant derivatives. The boxed sequence in the T.brucei U1 snRNA indicates the Sm site; the two arrows indicate a potential second stem–loop. Below, the sequences of the stem–loop derivatives are given; the circled nucleotides mark the two positions in the human loop that differ from the T.brucei sequence, and the single-nucleotide mutation (A21) in the mutant human loop.

Techniques Used: In Vitro, Sequencing, Western Blot, Affinity Purification, SDS Page, Molecular Weight, Purification, End Labeling, Labeling, Mutagenesis, Incubation

15) Product Images from "Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics"

Article Title: Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060333

CD63 participates in HCV entry through a direct interaction with HCV E2. (A) Co-immunoprecipitations of CD63 and CD81 by HCV E2. Extracts of Huh 7.5.1 cells, which were infected with JC1 E2 FLAG virus (MOI 0.3) for 72 hrs, were analyzed by Western blotting to detect the indicated proteins before and after immunoprecipitations with a FLAG antibody or a control mouse antibody. Actin is a negative control. (B) GST pull-down assays with purified proteins. GST-fused CD63 EC2 (GST-CD63) and GST-fused CD81 LEL (GST-CD81) proteins were expressed in E. coli and then purified ( Methods ). FLAG-tagged HCV E2 (FLAG-E2) proteins were expressed in yeast and then purified ( Methods ). After incubating the purified FLAG-E2 proteins with GST, GST-CD81 or GST-CD63 proteins for 2 hrs at 4°C, GST, GST-fusion proteins, and their associated proteins were precipitated with GSH Sepharose 4B. The resin-bound proteins were analyzed by Western blotting with antibodies against GST or FLAG. Degraded forms of GST-CD63 and GST-CD81 (indicated by asterisks) were also precipitated by the GSH resin. (C) Effect of a polypeptide corresponding to the CD63 EC2 domain on HCV infection. JFH1 5A-Rluc virus was incubated with GST or GST-CD63 for 2 hrs at 4°C. Huh7.5.1 cells were then inoculated with the virus–polypeptide mixtures by incubating for 3 hrs at 37°C, and the cells were further cultivated for 48 hrs. Virus infectivity was monitored by measuring Renilla luciferase activities in cell extracts, and normalized to the amounts of proteins in cell extracts (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted. (D) Effect of an anti-CD63 antibody (BEM-1 from Santa Cruz Biotechnologies) on HCV infection. Huh7.5.1 cells were pre-incubated with a negative control mouse IgG1, a positive control anti-CD81 antibody, or an anti-CD63 antibody at the indicated concentrations for 1 hour at 37°C, and then inoculated with JFH1 5A-Rluc virus (MOI of 0.3). The cells were cultivated for additional 48 hrs, and then Renilla luciferase activities in cell lysates were measured and normalized to the amounts of proteins in lysates (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted.
Figure Legend Snippet: CD63 participates in HCV entry through a direct interaction with HCV E2. (A) Co-immunoprecipitations of CD63 and CD81 by HCV E2. Extracts of Huh 7.5.1 cells, which were infected with JC1 E2 FLAG virus (MOI 0.3) for 72 hrs, were analyzed by Western blotting to detect the indicated proteins before and after immunoprecipitations with a FLAG antibody or a control mouse antibody. Actin is a negative control. (B) GST pull-down assays with purified proteins. GST-fused CD63 EC2 (GST-CD63) and GST-fused CD81 LEL (GST-CD81) proteins were expressed in E. coli and then purified ( Methods ). FLAG-tagged HCV E2 (FLAG-E2) proteins were expressed in yeast and then purified ( Methods ). After incubating the purified FLAG-E2 proteins with GST, GST-CD81 or GST-CD63 proteins for 2 hrs at 4°C, GST, GST-fusion proteins, and their associated proteins were precipitated with GSH Sepharose 4B. The resin-bound proteins were analyzed by Western blotting with antibodies against GST or FLAG. Degraded forms of GST-CD63 and GST-CD81 (indicated by asterisks) were also precipitated by the GSH resin. (C) Effect of a polypeptide corresponding to the CD63 EC2 domain on HCV infection. JFH1 5A-Rluc virus was incubated with GST or GST-CD63 for 2 hrs at 4°C. Huh7.5.1 cells were then inoculated with the virus–polypeptide mixtures by incubating for 3 hrs at 37°C, and the cells were further cultivated for 48 hrs. Virus infectivity was monitored by measuring Renilla luciferase activities in cell extracts, and normalized to the amounts of proteins in cell extracts (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted. (D) Effect of an anti-CD63 antibody (BEM-1 from Santa Cruz Biotechnologies) on HCV infection. Huh7.5.1 cells were pre-incubated with a negative control mouse IgG1, a positive control anti-CD81 antibody, or an anti-CD63 antibody at the indicated concentrations for 1 hour at 37°C, and then inoculated with JFH1 5A-Rluc virus (MOI of 0.3). The cells were cultivated for additional 48 hrs, and then Renilla luciferase activities in cell lysates were measured and normalized to the amounts of proteins in lysates (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted.

Techniques Used: Infection, Western Blot, Negative Control, Purification, Incubation, Luciferase, Positive Control

16) Product Images from "Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production"

Article Title: Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production

Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

doi: 10.2183/pjab.87.563

NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.
Figure Legend Snippet: NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.

Techniques Used: In Vitro, Ubiquitin Assay, Expressing, Plasmid Preparation, Transfection, Incubation, Over Expression

17) Product Images from "DAZAP1 regulates the splicing of Crem, Crisp2 and Pot1a transcripts"

Article Title: DAZAP1 regulates the splicing of Crem, Crisp2 and Pot1a transcripts

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt746

EMSA assays on DAZAP1-RNA binding. ( A ) Sequences of the RNA probes from the three DAZAP1 target genes. Deleted regions are indicated with doted lines. ( B ) EMSA gel patterns. 32 P-labelled probes were incubated with buffer only (−) or 500 nM GST-DAZAP1 (+) and analysed by 5% native PAGE. Determination of the dissociation constants of the binding between DAZAP1 and the various RNA probes is shown in Supplementary Figure S4 .
Figure Legend Snippet: EMSA assays on DAZAP1-RNA binding. ( A ) Sequences of the RNA probes from the three DAZAP1 target genes. Deleted regions are indicated with doted lines. ( B ) EMSA gel patterns. 32 P-labelled probes were incubated with buffer only (−) or 500 nM GST-DAZAP1 (+) and analysed by 5% native PAGE. Determination of the dissociation constants of the binding between DAZAP1 and the various RNA probes is shown in Supplementary Figure S4 .

Techniques Used: RNA Binding Assay, Incubation, Clear Native PAGE, Binding Assay

Mapping DAZAP1 binding sites and splicing regulatory regions in the target genes. ( A ) Crem , ( B ) Crisp2 and ( C ) Pot1a . For each section dedicated to a specific gene, the sequence of the regulated exon (in Capital bold letters) and its flanking intronic regions (in low case) is shown at top. EMSA probes (P1–P5) are indicated with underlying double-headed arrows, and segments deleted in the various minigene deletion constructs (A– E ) are boxed. The left panel below the sequence shows the EMSA gel patterns. 32 P-labelled probes, indicated at top, were incubated with buffer only (mock), GST, or GST-DAZAP1 and analysed by 5% native PAGE. The middle panel shows RT-PCR gel patterns of splicing assays of the various minigene deletion constructs. The PSI of each reaction is shown below the gel lane. The right panel shows the relative change of PSI by DAZAP1, calculated from the middle panel by dividing the PSI value in the presence of exogenous DAZAP1 with that in the absence of exogenous DAZAP1. Three independent experiments were performed, and statistical significance of the differences was calculated using the paired t test. * P
Figure Legend Snippet: Mapping DAZAP1 binding sites and splicing regulatory regions in the target genes. ( A ) Crem , ( B ) Crisp2 and ( C ) Pot1a . For each section dedicated to a specific gene, the sequence of the regulated exon (in Capital bold letters) and its flanking intronic regions (in low case) is shown at top. EMSA probes (P1–P5) are indicated with underlying double-headed arrows, and segments deleted in the various minigene deletion constructs (A– E ) are boxed. The left panel below the sequence shows the EMSA gel patterns. 32 P-labelled probes, indicated at top, were incubated with buffer only (mock), GST, or GST-DAZAP1 and analysed by 5% native PAGE. The middle panel shows RT-PCR gel patterns of splicing assays of the various minigene deletion constructs. The PSI of each reaction is shown below the gel lane. The right panel shows the relative change of PSI by DAZAP1, calculated from the middle panel by dividing the PSI value in the presence of exogenous DAZAP1 with that in the absence of exogenous DAZAP1. Three independent experiments were performed, and statistical significance of the differences was calculated using the paired t test. * P

Techniques Used: Binding Assay, Sequencing, Construct, Incubation, Clear Native PAGE, Reverse Transcription Polymerase Chain Reaction

18) Product Images from "KCTD1 Suppresses Canonical Wnt Signaling Pathway by Enhancing ?-catenin Degradation"

Article Title: KCTD1 Suppresses Canonical Wnt Signaling Pathway by Enhancing ?-catenin Degradation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0094343

Identification of β-catenin binding domain in KCTD1. (A) Schematic representation of KCTD1 domains, deletion and mutant constructs used for pull-down analysis and luciferase assays. And the five silent mutations in the siRNA-target sequence of wild-type KCTD1 cDNA were shown, the resulting protein mutKCTD1 confers resistance to the siRNA and no change in the amino acid sequence compared with the wild-type. (B) The full-length and truncated proteins of GST-KCTD1 were bacterially expressed, purified and detected with Western blots using mouse monoclonal anti-GST antibodies. (C) GST pull-down experiments were performed with GST, GST fusion proteins above and His-β-catenin recombinant proteins analyzed by immunoblots with mouse monoclonal antibodies against His-tag.
Figure Legend Snippet: Identification of β-catenin binding domain in KCTD1. (A) Schematic representation of KCTD1 domains, deletion and mutant constructs used for pull-down analysis and luciferase assays. And the five silent mutations in the siRNA-target sequence of wild-type KCTD1 cDNA were shown, the resulting protein mutKCTD1 confers resistance to the siRNA and no change in the amino acid sequence compared with the wild-type. (B) The full-length and truncated proteins of GST-KCTD1 were bacterially expressed, purified and detected with Western blots using mouse monoclonal anti-GST antibodies. (C) GST pull-down experiments were performed with GST, GST fusion proteins above and His-β-catenin recombinant proteins analyzed by immunoblots with mouse monoclonal antibodies against His-tag.

Techniques Used: Binding Assay, Mutagenesis, Construct, Luciferase, Sequencing, Purification, Western Blot, Recombinant

Identification of KCTD1 binding domain in β-catenin. (A) Schematic representation of protein domain structure of β-catenin and its deletion constructs used for pull-down and luciferase assays. The mutant phosphorylation sites (the Ser45 and Ser33/37/Thr41 sites) in β-catenin mutations were shown. (B) Bacterially expressed and purified His-β-catenin fusion proteins were detected with Western blots using mouse monoclonal antibodies against His-tag. (C) His-tag pull-down experiments were performed with GST-KCTD1 and the full-length or truncated proteins of His-β-catenin analyzed by Western blots using mouse monoclonal anti-GST antibodies. GST proteins were used as negative control.
Figure Legend Snippet: Identification of KCTD1 binding domain in β-catenin. (A) Schematic representation of protein domain structure of β-catenin and its deletion constructs used for pull-down and luciferase assays. The mutant phosphorylation sites (the Ser45 and Ser33/37/Thr41 sites) in β-catenin mutations were shown. (B) Bacterially expressed and purified His-β-catenin fusion proteins were detected with Western blots using mouse monoclonal antibodies against His-tag. (C) His-tag pull-down experiments were performed with GST-KCTD1 and the full-length or truncated proteins of His-β-catenin analyzed by Western blots using mouse monoclonal anti-GST antibodies. GST proteins were used as negative control.

Techniques Used: Binding Assay, Construct, Luciferase, Mutagenesis, Purification, Western Blot, Negative Control

19) Product Images from "Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)"

Article Title: Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl1166

( A ) Interaction between hEXO1(K418A) and hMLH1/hMSH2. Lane 1: beads + hEXO1(K418A); 2: E. coli lysate + IVTT hEXO1(K418A); 3: GST-hMSH2 + vector; 4: GST-hMLH1 + vector; 5: GST-hMSH2 + hEXO1(K418A); 6: GST-hMSH2 + hEXO1; 7: GST-hMLH1 + hEXO1(K418A); 8: GST-hMLH1 + hEXO1; 9: hEXO1(K418A); 10: hEXO1. ( B ) Interaction between hEXO1(K418A) and importin α3. Lane 1: hEXO1(K418A); 2: beads + hEXO1(K418A); 3: α3/β + hEXO1(K418A); 4: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:0.5); 5: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:1); 6: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:2); 7: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:3); 8: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:5); 9: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:10); 10: hEXO1; 11: beads + hEXO1; 12: α3/β + hEXO1; 13: α3/β + hEXO1 + unlabeled hEXO1 (1:0.5); 14: α3/β + hEXO1 + unlabeled hEXO1 (1:1); 15: α3/β + hEXO1 + unlabeled hEXO1 (1:2); 16: α3/β + hEXO1 + unlabeled hEXO1 (1:3); 17: α3/β + hEXO1 + unlabeled hEXO1 (1:5); 18: α3/β + hEXO1 + unlabeled hEXO1 (1:10). ( C ) Relative intensity of bands shown in (B) as a function of unlabeled IVTT-hEXO1/labelled IVTT-hEXO1/hEXO1(K418A). Open circles: labeled hEXO1 competed with excess unlabeled hEXO1; closed circles: labeled hEXO1(K418A) competed with excess unlabeled hEXO1. Standard deviations are shown. Each data point in this figure represents the average from three independent experiments.
Figure Legend Snippet: ( A ) Interaction between hEXO1(K418A) and hMLH1/hMSH2. Lane 1: beads + hEXO1(K418A); 2: E. coli lysate + IVTT hEXO1(K418A); 3: GST-hMSH2 + vector; 4: GST-hMLH1 + vector; 5: GST-hMSH2 + hEXO1(K418A); 6: GST-hMSH2 + hEXO1; 7: GST-hMLH1 + hEXO1(K418A); 8: GST-hMLH1 + hEXO1; 9: hEXO1(K418A); 10: hEXO1. ( B ) Interaction between hEXO1(K418A) and importin α3. Lane 1: hEXO1(K418A); 2: beads + hEXO1(K418A); 3: α3/β + hEXO1(K418A); 4: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:0.5); 5: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:1); 6: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:2); 7: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:3); 8: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:5); 9: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:10); 10: hEXO1; 11: beads + hEXO1; 12: α3/β + hEXO1; 13: α3/β + hEXO1 + unlabeled hEXO1 (1:0.5); 14: α3/β + hEXO1 + unlabeled hEXO1 (1:1); 15: α3/β + hEXO1 + unlabeled hEXO1 (1:2); 16: α3/β + hEXO1 + unlabeled hEXO1 (1:3); 17: α3/β + hEXO1 + unlabeled hEXO1 (1:5); 18: α3/β + hEXO1 + unlabeled hEXO1 (1:10). ( C ) Relative intensity of bands shown in (B) as a function of unlabeled IVTT-hEXO1/labelled IVTT-hEXO1/hEXO1(K418A). Open circles: labeled hEXO1 competed with excess unlabeled hEXO1; closed circles: labeled hEXO1(K418A) competed with excess unlabeled hEXO1. Standard deviations are shown. Each data point in this figure represents the average from three independent experiments.

Techniques Used: Plasmid Preparation, Labeling

20) Product Images from "Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair *Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair * S⃞"

Article Title: Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair *Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M808906200

CtIP mutations affect DSB processing. A , cells expressing CtIP variants were treated with DMSO (-) or 25 μ m roscovitine ( Rosc. )(+) and then irradiated with 10 Gy of IR. One h later, cells were immunostained for RPA or γH2AX. Averages and standard deviations ( error bars ) of three independent experiments are shown. At least 200 cells were counted per experiment. B , representative images of cells treated in A. C , the number of RPA foci per cell in cells expressing the GFP-CtIP-T847E mutant in the presence or absence of the CDK inhibitor roscovitine. Error bars , standard deviations. D , an immunoblot of protein extracts, collected 1 h after irradiation (10 Gy), of cells expressing the indicated GFP-CtIP fusions. Panels to the left and right contain samples derived from cells treated in the absence or presence of roscovitine, respectively.
Figure Legend Snippet: CtIP mutations affect DSB processing. A , cells expressing CtIP variants were treated with DMSO (-) or 25 μ m roscovitine ( Rosc. )(+) and then irradiated with 10 Gy of IR. One h later, cells were immunostained for RPA or γH2AX. Averages and standard deviations ( error bars ) of three independent experiments are shown. At least 200 cells were counted per experiment. B , representative images of cells treated in A. C , the number of RPA foci per cell in cells expressing the GFP-CtIP-T847E mutant in the presence or absence of the CDK inhibitor roscovitine. Error bars , standard deviations. D , an immunoblot of protein extracts, collected 1 h after irradiation (10 Gy), of cells expressing the indicated GFP-CtIP fusions. Panels to the left and right contain samples derived from cells treated in the absence or presence of roscovitine, respectively.

Techniques Used: Expressing, Irradiation, Recombinase Polymerase Amplification, Mutagenesis, Derivative Assay

Functional effects of mutating Thr-847 of CtIP. A , alignment of the region conserved among Sae2/CtIP orthologues. Arrows show the position of the conserved CtIP Thr-847 and Sae2 Ser-267. A. thaliana, Arabidopsis thaliana ; C. elegans, Caenorhabditis elegans ; P. nodorum, Phaeosphaeria nodorum ; C. globosum, Chaetomium globosum ; N. crassa, Neurospora crassa ; C. neoformans, Cryptococcus neoformans ; Y. lipolytica, Yarrowia lipolytica ; A. gossypii, Ashbya gossypii. B , expression levels of GFP-CtIP derivatives in stably transfected clones before ( left ) or after ( right ) siRNA depletion of endogenous CtIP ( siCtIP ). C , representative confocal microscope images of cells expressing wild-type or T847A CtIP variants after immunostaining with a phospho-specific antibody raised against phosphorylated Thr-847. Cells were incubated in the presence of the CDK inhibitor roscovitine where indicated. D , a GST-fused wild-type or T847A mutant CtIP C-terminal fragment (residues 750-897) was affinity-purified with glutathione-Sepharose 4B and then incubated with [γ- 32 P]ATP in the presence or absence of recombinant CDK2/cyclin A, separated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. Proteins were detected with an anti-GST antibody ( bottom ), and phosphorylation was visualized by autoradiography (CDK assay; top ).
Figure Legend Snippet: Functional effects of mutating Thr-847 of CtIP. A , alignment of the region conserved among Sae2/CtIP orthologues. Arrows show the position of the conserved CtIP Thr-847 and Sae2 Ser-267. A. thaliana, Arabidopsis thaliana ; C. elegans, Caenorhabditis elegans ; P. nodorum, Phaeosphaeria nodorum ; C. globosum, Chaetomium globosum ; N. crassa, Neurospora crassa ; C. neoformans, Cryptococcus neoformans ; Y. lipolytica, Yarrowia lipolytica ; A. gossypii, Ashbya gossypii. B , expression levels of GFP-CtIP derivatives in stably transfected clones before ( left ) or after ( right ) siRNA depletion of endogenous CtIP ( siCtIP ). C , representative confocal microscope images of cells expressing wild-type or T847A CtIP variants after immunostaining with a phospho-specific antibody raised against phosphorylated Thr-847. Cells were incubated in the presence of the CDK inhibitor roscovitine where indicated. D , a GST-fused wild-type or T847A mutant CtIP C-terminal fragment (residues 750-897) was affinity-purified with glutathione-Sepharose 4B and then incubated with [γ- 32 P]ATP in the presence or absence of recombinant CDK2/cyclin A, separated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. Proteins were detected with an anti-GST antibody ( bottom ), and phosphorylation was visualized by autoradiography (CDK assay; top ).

Techniques Used: Functional Assay, Expressing, Stable Transfection, Transfection, Clone Assay, Microscopy, Immunostaining, Incubation, Mutagenesis, Affinity Purification, Recombinant, SDS Page, Autoradiography

21) Product Images from "CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex"

Article Title: CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200405094

The COOH-terminal domain of CLASP2 is responsible for association with the cell cortex and Golgi complex. (A–F) TIRF microscopy images of live HeLa cells, expressing GFP-CLASP1α (A), GFP-CLASP2γ (B), GFP-α-tubulin (C and D), or EB3-GFP (E and F). Cells were either not treated with siRNAs (A and B), or treated for 72 h with control (C and E) or CLASP1+2#B siRNAs (D and F). The contrast is inverted. Bars, 10 μm. (G) Schematic representation of CLASP2γ and the relevant deletion mutants. (H) HeLa cells were transfected with GFP-CLASP2-C and stained for the Golgi marker GM130. Bar, 10 μm. (I) HeLa cells were transfected with GFP-CLASP2 or GFP-CLASP2-ΔC and were either fixed directly or treated with 10 μM nocodazole for 1 h before fixation and stained for α-tubulin. Bars, 10 μm.
Figure Legend Snippet: The COOH-terminal domain of CLASP2 is responsible for association with the cell cortex and Golgi complex. (A–F) TIRF microscopy images of live HeLa cells, expressing GFP-CLASP1α (A), GFP-CLASP2γ (B), GFP-α-tubulin (C and D), or EB3-GFP (E and F). Cells were either not treated with siRNAs (A and B), or treated for 72 h with control (C and E) or CLASP1+2#B siRNAs (D and F). The contrast is inverted. Bars, 10 μm. (G) Schematic representation of CLASP2γ and the relevant deletion mutants. (H) HeLa cells were transfected with GFP-CLASP2-C and stained for the Golgi marker GM130. Bar, 10 μm. (I) HeLa cells were transfected with GFP-CLASP2 or GFP-CLASP2-ΔC and were either fixed directly or treated with 10 μM nocodazole for 1 h before fixation and stained for α-tubulin. Bars, 10 μm.

Techniques Used: Microscopy, Expressing, Transfection, Staining, Marker

22) Product Images from "Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration *Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration * S⃞"

Article Title: Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration *Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M800617200

SAM domain deleted DLC-1 shows enhanced catalytic activity for RhoA. A , bacterially expressed full-length, SAM domain deleted (amino acids 77-1091), and RhoGAP domain fragment (amino acids 609-878) of DLC-1 were purified for analysis of in vitro GAP activity. Purified GST-RhoA fusion proteins were preloaded with GTP, and GTP hydrolysis was monitored by incubation with a phosphate-binding protein that undergoes a major increase in fluorescence upon binding inorganic phosphate. B , GTP hydrolysis activities of DLC-1 constructs. C , GTP loading of RhoA in cells was monitored by a Rhotekin pull-down assay as described under “Experimental Procedures.”
Figure Legend Snippet: SAM domain deleted DLC-1 shows enhanced catalytic activity for RhoA. A , bacterially expressed full-length, SAM domain deleted (amino acids 77-1091), and RhoGAP domain fragment (amino acids 609-878) of DLC-1 were purified for analysis of in vitro GAP activity. Purified GST-RhoA fusion proteins were preloaded with GTP, and GTP hydrolysis was monitored by incubation with a phosphate-binding protein that undergoes a major increase in fluorescence upon binding inorganic phosphate. B , GTP hydrolysis activities of DLC-1 constructs. C , GTP loading of RhoA in cells was monitored by a Rhotekin pull-down assay as described under “Experimental Procedures.”

Techniques Used: Activity Assay, Purification, In Vitro, Incubation, Binding Assay, Fluorescence, Construct, Pull Down Assay

23) Product Images from "Temporal and Spatial Regulation of the Phosphatidate Phosphatases Lipin 1 and 2 *Temporal and Spatial Regulation of the Phosphatidate Phosphatases Lipin 1 and 2 * S⃞Temporal and Spatial Regulation of the Phosphatidate Phosphatases Lipin 1 and 2 * S⃞ "

Article Title: Temporal and Spatial Regulation of the Phosphatidate Phosphatases Lipin 1 and 2 *Temporal and Spatial Regulation of the Phosphatidate Phosphatases Lipin 1 and 2 * S⃞Temporal and Spatial Regulation of the Phosphatidate Phosphatases Lipin 1 and 2 * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M804278200

Subcellular localization of lipin 1 and 2. A , localization of lipin 1-HA and lipin 2-HA fusions in formaldehyde fixed HeLa M cells. Expression of lipin-HA fusions was driven by the cytomegalovirus promoter. Bars , 5 μm. B , localization of lipin 1-GFP and lipin 2-GFP fusions in live HeLa M cells. Live HeLa M cells transiently expressing a dsRed-ER reporter and lipin 1-GFP ( upper panel ) or lipin 2-GFP ( lower panel ) were visualized by confocal microscopy. Expression of the tagged lipins was driven by the cytomegalovirus promoter. Bars , 5 μm. C , fractionation of endogenous lipin 1 and 2 in HeLa M cells. The cells were lysed in either buffer alone or buffer containing 1 m NaCl, 1% Triton X-100, or both. The extracts were incubated for 20 min at 4 °C and then centrifuged at 100,000 × g for 1 h. Equal volumes from the pellet ( P ) and supernatant ( S ) were loaded on an 8% SDS-PAGE and immunoblotted using the indicated antibodies.
Figure Legend Snippet: Subcellular localization of lipin 1 and 2. A , localization of lipin 1-HA and lipin 2-HA fusions in formaldehyde fixed HeLa M cells. Expression of lipin-HA fusions was driven by the cytomegalovirus promoter. Bars , 5 μm. B , localization of lipin 1-GFP and lipin 2-GFP fusions in live HeLa M cells. Live HeLa M cells transiently expressing a dsRed-ER reporter and lipin 1-GFP ( upper panel ) or lipin 2-GFP ( lower panel ) were visualized by confocal microscopy. Expression of the tagged lipins was driven by the cytomegalovirus promoter. Bars , 5 μm. C , fractionation of endogenous lipin 1 and 2 in HeLa M cells. The cells were lysed in either buffer alone or buffer containing 1 m NaCl, 1% Triton X-100, or both. The extracts were incubated for 20 min at 4 °C and then centrifuged at 100,000 × g for 1 h. Equal volumes from the pellet ( P ) and supernatant ( S ) were loaded on an 8% SDS-PAGE and immunoblotted using the indicated antibodies.

Techniques Used: Expressing, Confocal Microscopy, Fractionation, Incubation, SDS Page

Depletion of lipin 1 and 2 in HeLa M cells. A , siRNA down-regulates lipin 1 and 2 mRNA and protein expression. The cells were transfected with either a nontargeting ( Control ), or lipin 1, lipin 2, or lipin 1 and 2 small interfering RNA duplexes. 72 h after transfection, mRNA ( left panel ) or protein ( right panel ) levels were determined by real time PCR and immunoblotting, respectively, using the indicated antibodies. B , cell cycle profiles of cells from A were determined by flow cytometry as described under “Experimental Procedures.” The significance of the difference between control and lipin 1 siRNA-treated cells found in the G 1 phase is indicated by p
Figure Legend Snippet: Depletion of lipin 1 and 2 in HeLa M cells. A , siRNA down-regulates lipin 1 and 2 mRNA and protein expression. The cells were transfected with either a nontargeting ( Control ), or lipin 1, lipin 2, or lipin 1 and 2 small interfering RNA duplexes. 72 h after transfection, mRNA ( left panel ) or protein ( right panel ) levels were determined by real time PCR and immunoblotting, respectively, using the indicated antibodies. B , cell cycle profiles of cells from A were determined by flow cytometry as described under “Experimental Procedures.” The significance of the difference between control and lipin 1 siRNA-treated cells found in the G 1 phase is indicated by p

Techniques Used: Expressing, Transfection, Small Interfering RNA, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

Mitotic phosphorylation of lipin 1 and 2 regulates their PAP1 activity. A , lipins are phosphorylated on Cdk1 motifs during mitosis. Left panel , Endogenous lipin 1 and lipin 2 were immunoprecipitated ( IP ) from extracts of asynchronous ( Asy ) or mitotic HeLa M cells synchronized either by nocodazole arrest ( Noc ) or double thymidine arrest at the G 1 /S boundary followed by release and collection during mitosis (8–10 h post-release) ( D. Thy ). Immune pellets were analyzed by Western blotting using anti-lipin 1, anti-lipin 2, and anti-phospho-Ser/Thr-Pro (MPM2) antibodies. Right panel , flow cytometry of the cells used for the lipin immunoprecipitations. B , mitotic phosphorylation of lipins inhibits their PAP1 activity. Upper panel , immunoprecipitated lipin 1 and 2 from asynchronous or nocodazole-treated HeLa M cells with or without incubation with λ-phosphatase (λ PPase ) were analyzed by Western blotting as indicated. Mock immunoprecipitations were performed using the preimmune lipin 1 and 2 sera. Lower panel , immune pellets from the above immunoprecipitations were assayed for PAP1 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. The data for the asynchronous and nocodazole samples are the averages from two independent experiments. C , total PAP1 activity decreases during mitosis. Lysates from asynchronous or mitotic cells were assayed for PAP1 and PAP2 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. D , mitotic phosphorylation of lipins regulates their membrane recruitment. Lysates from asynchronous or mitotic cells were centrifuged at 100,000 × g . Equal volumes from the pellet ( P ) and supernatant ( S ) were immunoblotted using the indicated antibodies.
Figure Legend Snippet: Mitotic phosphorylation of lipin 1 and 2 regulates their PAP1 activity. A , lipins are phosphorylated on Cdk1 motifs during mitosis. Left panel , Endogenous lipin 1 and lipin 2 were immunoprecipitated ( IP ) from extracts of asynchronous ( Asy ) or mitotic HeLa M cells synchronized either by nocodazole arrest ( Noc ) or double thymidine arrest at the G 1 /S boundary followed by release and collection during mitosis (8–10 h post-release) ( D. Thy ). Immune pellets were analyzed by Western blotting using anti-lipin 1, anti-lipin 2, and anti-phospho-Ser/Thr-Pro (MPM2) antibodies. Right panel , flow cytometry of the cells used for the lipin immunoprecipitations. B , mitotic phosphorylation of lipins inhibits their PAP1 activity. Upper panel , immunoprecipitated lipin 1 and 2 from asynchronous or nocodazole-treated HeLa M cells with or without incubation with λ-phosphatase (λ PPase ) were analyzed by Western blotting as indicated. Mock immunoprecipitations were performed using the preimmune lipin 1 and 2 sera. Lower panel , immune pellets from the above immunoprecipitations were assayed for PAP1 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. The data for the asynchronous and nocodazole samples are the averages from two independent experiments. C , total PAP1 activity decreases during mitosis. Lysates from asynchronous or mitotic cells were assayed for PAP1 and PAP2 activity as described under “Experimental Procedures.” The results shown were determined from triplicate enzyme assays ± S.D. D , mitotic phosphorylation of lipins regulates their membrane recruitment. Lysates from asynchronous or mitotic cells were centrifuged at 100,000 × g . Equal volumes from the pellet ( P ) and supernatant ( S ) were immunoblotted using the indicated antibodies.

Techniques Used: Activity Assay, Immunoprecipitation, Western Blot, Flow Cytometry, Cytometry, Incubation

Reciprocal pattern of lipin 1 and 2 protein expression during adipogenesis. A , 3T3-L1 preadipocytes were induced to differentiate for 12 days. At the indicated time points, the cell lysates were prepared, protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using lipin 1 and 2 antibodies. Adipocyte differentiation was monitored by following αP2 expression. B , lipin 2 is phosphorylated in 3T3-L1 cells. Endogenous lipin 2 was immunoprecipitated ( IP ) from extracts of 3T3-L1 preadipocytes. Immune pellets were incubated with or without λ-phosphatase (λ PPase ) and analyzed by SDS-PAGE followed by immunoblotting using lipin 2 antibodies.
Figure Legend Snippet: Reciprocal pattern of lipin 1 and 2 protein expression during adipogenesis. A , 3T3-L1 preadipocytes were induced to differentiate for 12 days. At the indicated time points, the cell lysates were prepared, protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using lipin 1 and 2 antibodies. Adipocyte differentiation was monitored by following αP2 expression. B , lipin 2 is phosphorylated in 3T3-L1 cells. Endogenous lipin 2 was immunoprecipitated ( IP ) from extracts of 3T3-L1 preadipocytes. Immune pellets were incubated with or without λ-phosphatase (λ PPase ) and analyzed by SDS-PAGE followed by immunoblotting using lipin 2 antibodies.

Techniques Used: Expressing, Bradford Assay, SDS Page, Immunoprecipitation, Incubation

The function of lipin 1 and 2 is evolutionarily conserved from yeast to mammals. A , primary structure of Pah1p, lipin 1, and lipin 2. The conserved N-terminal domain and the C-terminal PAP domain are indicated. B , lipin 2 rescues the temperature-sensitive growth defect of the pah1 Δ mutant. pah1 Δ cells were transformed with a 2 μ (high copy) vector containing the indicated genes, spotted on YEPD plates, and grown at 30 or 37 °C for 36 h. C , lipins can rescue the nup84 Δ spo7 Δ synthetic lethality. The nup84 Δ spo7 Δ double deletion strain carrying a URA3 -containing vector expressing NUP84 was transformed with the indicated plasmids. Transformants were grown on plates containing 5-fluoroorotic acid for 3 days. No growth indicates synthetic lethality. D , lipin 2 rescues the nuclear structure defects of the pah1 Δ cells. The pah1 Δ mutant expressing an intranuclear reporter ( PUS1 -GFP) was transformed with the same plasmids as in B and visualized by confocal microscopy. Arrows point to cells containing irregularly shaped/multilobed nuclei. The percentage of cells containing a single round nucleus is given. Two different transformants per strain were analyzed and for each one the number of cells counted was 200. Bars , 5 μm. E , expression of lipin 1 and lipin 2 in yeast. Protein extracts from pah1 Δ cells expressing PAH1 -GFP, lipin 1-GFP or lipin 2-GFP fusions and grown at 30 °C to early logarithmic phase were analyzed by Western blot using anti-GFP antibodies.
Figure Legend Snippet: The function of lipin 1 and 2 is evolutionarily conserved from yeast to mammals. A , primary structure of Pah1p, lipin 1, and lipin 2. The conserved N-terminal domain and the C-terminal PAP domain are indicated. B , lipin 2 rescues the temperature-sensitive growth defect of the pah1 Δ mutant. pah1 Δ cells were transformed with a 2 μ (high copy) vector containing the indicated genes, spotted on YEPD plates, and grown at 30 or 37 °C for 36 h. C , lipins can rescue the nup84 Δ spo7 Δ synthetic lethality. The nup84 Δ spo7 Δ double deletion strain carrying a URA3 -containing vector expressing NUP84 was transformed with the indicated plasmids. Transformants were grown on plates containing 5-fluoroorotic acid for 3 days. No growth indicates synthetic lethality. D , lipin 2 rescues the nuclear structure defects of the pah1 Δ cells. The pah1 Δ mutant expressing an intranuclear reporter ( PUS1 -GFP) was transformed with the same plasmids as in B and visualized by confocal microscopy. Arrows point to cells containing irregularly shaped/multilobed nuclei. The percentage of cells containing a single round nucleus is given. Two different transformants per strain were analyzed and for each one the number of cells counted was 200. Bars , 5 μm. E , expression of lipin 1 and lipin 2 in yeast. Protein extracts from pah1 Δ cells expressing PAH1 -GFP, lipin 1-GFP or lipin 2-GFP fusions and grown at 30 °C to early logarithmic phase were analyzed by Western blot using anti-GFP antibodies.

Techniques Used: Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Confocal Microscopy, Western Blot

shRNA-mediated silencing of lipin 1 and 2 in differentiating adipocytes. 3T3-L1 preadipocytes stably transfected with retroviral vectors expressing shRNA targeting lipin 1 ( Lipin 1 ), lipin 2 ( Lipin 2 ) or a control sequence ( Control ), were induced to differentiate for 8 days. At the indicated time points, the cell lysates were prepared, the protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies.
Figure Legend Snippet: shRNA-mediated silencing of lipin 1 and 2 in differentiating adipocytes. 3T3-L1 preadipocytes stably transfected with retroviral vectors expressing shRNA targeting lipin 1 ( Lipin 1 ), lipin 2 ( Lipin 2 ) or a control sequence ( Control ), were induced to differentiate for 8 days. At the indicated time points, the cell lysates were prepared, the protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies.

Techniques Used: shRNA, Stable Transfection, Transfection, Expressing, Sequencing, Bradford Assay, SDS Page

24) Product Images from "Rab11A-Controlled Assembly of the Inner Membrane Complex Is Required for Completion of Apicomplexan Cytokinesis"

Article Title: Rab11A-Controlled Assembly of the Inner Membrane Complex Is Required for Completion of Apicomplexan Cytokinesis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000270

PfRab11A associates with PfMTIP in vitro . The purified recombinant proteins PfRab11A-His and PfRab5C-His (top panel) were mixed with GST or GST–MTIP (bottom panel). Anti-His antibodies (Santa Cruz) were used to detect pulled-down His-tagged Rab protein and only PfRab11A associates with PfMTIP. The upper panel of the pull down correspond to Ponceau S stain of the membrane to demonstrate loadings of GST and GST-MTIP, respectively.
Figure Legend Snippet: PfRab11A associates with PfMTIP in vitro . The purified recombinant proteins PfRab11A-His and PfRab5C-His (top panel) were mixed with GST or GST–MTIP (bottom panel). Anti-His antibodies (Santa Cruz) were used to detect pulled-down His-tagged Rab protein and only PfRab11A associates with PfMTIP. The upper panel of the pull down correspond to Ponceau S stain of the membrane to demonstrate loadings of GST and GST-MTIP, respectively.

Techniques Used: In Vitro, Purification, Recombinant, Staining

25) Product Images from "Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival"

Article Title: Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

Journal: PLoS ONE

doi: 10.1371/journal.pone.0007254

HPV-16 E7 influences the balance between oxidized and reduced GSTP1. A: Western blots for GSTP1 after non-reducing SDS-PAGE in control and in HPV-16 E7-infected HaCaT cells under normal conditions (untreated) or after induction of oxidative stress by exposure to UV (UVB, 5 mJ/cm 2 for 1 min) or hydrogen peroxide (0.5 mM H 2 O 2 for 30 min). In the first case, detection was performed after a 90 min or 5 h recovery period, while in the second case detection was performed after a 6 h period. In all cases, HPV-16 E7 expression was accompanied by a drastic decrease in the GSTP1 oxidized multimeric form (GSTP1 multimer ox ) and of the lowest band, which represents the oxidized GSTP1 monomer (GSTP1 monomer ox ). GSTP1 monomer red denotes the reduced form. B: Western blots for GSTP1 after non-reducing SDS-PAGE in control, HPV-16 E7- and HPV-16 E7 Mut-infected HaCaT cells after exposure to UV (UVB, 5 mJ/cm 2 for 1 min) and 5 h recovery. The mutant oncoprotein appeared less efficient in protecting GSTP1 from oxidation. In both panels, blots were normalized against β-actin levels that were determined in matching gels run under reducing conditions.
Figure Legend Snippet: HPV-16 E7 influences the balance between oxidized and reduced GSTP1. A: Western blots for GSTP1 after non-reducing SDS-PAGE in control and in HPV-16 E7-infected HaCaT cells under normal conditions (untreated) or after induction of oxidative stress by exposure to UV (UVB, 5 mJ/cm 2 for 1 min) or hydrogen peroxide (0.5 mM H 2 O 2 for 30 min). In the first case, detection was performed after a 90 min or 5 h recovery period, while in the second case detection was performed after a 6 h period. In all cases, HPV-16 E7 expression was accompanied by a drastic decrease in the GSTP1 oxidized multimeric form (GSTP1 multimer ox ) and of the lowest band, which represents the oxidized GSTP1 monomer (GSTP1 monomer ox ). GSTP1 monomer red denotes the reduced form. B: Western blots for GSTP1 after non-reducing SDS-PAGE in control, HPV-16 E7- and HPV-16 E7 Mut-infected HaCaT cells after exposure to UV (UVB, 5 mJ/cm 2 for 1 min) and 5 h recovery. The mutant oncoprotein appeared less efficient in protecting GSTP1 from oxidation. In both panels, blots were normalized against β-actin levels that were determined in matching gels run under reducing conditions.

Techniques Used: Western Blot, SDS Page, Infection, Expressing, Mutagenesis

Roles of HPV-16 E7 and GSTP1 and their interaction in cell survival after UV exposure. A: After exposure to UV radiation (UVB, 5 mJ/cm 2 for 1 min), control and HPV-16 E7-expressing HaCaT cells showed a significant correlation between HPV-16 E7 expression and cell survival ( p = 0.000003, ***). Under the same conditions, GSTP1-deficient MCF-7 cells displayed no significant differences in survival, and thus no HPV-16 E7-related protection. Forced expression of GSTP1 in control and HPV-16 E7-infected MCF-7 cells restored the ability of HPV-16 E7 to protect against UV-induced cell death ( p = 0.0046, **). Values are the averages of three independent experiments performed in triplicate±SEM. B: Control, HPV-16 E7- and HPV-16 E7 Mut-infected HaCaT cells were transfected with non-targeting (NT) siRNA or with siRNA targeting GSTP1. After 72 h, GSTP1 siRNA-treated cells showed decreased GSTP1 protein expression. C: Cells, exposed to UV radiation as above, were collected and the number of Trypan blue-negative (live) cells was determined. The differential survival rate between cells unexposed and exposed to UV radiation was then calculated, and the results were expressed as percentages of dead cells after UV exposure. The results show a remarkable effect of HPV-16 E7 on cell survival (bars 1 vs. 3), a less effective protection by HPV-16 E7 Mut (bars 1 vs. 5), and the consequences of GSTP1 silencing in HPV-16 E7- (bars 3 vs. 4) and HPV-16 E7 Mut- expressing cells (bars 5 vs. 6). This panel indicates the role of GSTP1 in HPV-16 E7-induced cell survival and the importance of the binding ability of the oncoprotein in inducing the GSTP1-mediated increase in survival. Values are the average of three independent experiments performed in triplicate±SEM. Statistical significance of the reported values: bars 1 vs. 3 p = 0.00004, ***; bars 1 vs. 5 p = 0.0009, ***; bars 3 vs. 5 p = 0.000004, ***; bars 3 vs. 4 p = 0.0002, ***; bars 5 vs. 6 p = 0.0001, ***; bars 2 vs. 4 p = 0.0288, *; bars 4 vs. 6 p = 0.0007, ***.
Figure Legend Snippet: Roles of HPV-16 E7 and GSTP1 and their interaction in cell survival after UV exposure. A: After exposure to UV radiation (UVB, 5 mJ/cm 2 for 1 min), control and HPV-16 E7-expressing HaCaT cells showed a significant correlation between HPV-16 E7 expression and cell survival ( p = 0.000003, ***). Under the same conditions, GSTP1-deficient MCF-7 cells displayed no significant differences in survival, and thus no HPV-16 E7-related protection. Forced expression of GSTP1 in control and HPV-16 E7-infected MCF-7 cells restored the ability of HPV-16 E7 to protect against UV-induced cell death ( p = 0.0046, **). Values are the averages of three independent experiments performed in triplicate±SEM. B: Control, HPV-16 E7- and HPV-16 E7 Mut-infected HaCaT cells were transfected with non-targeting (NT) siRNA or with siRNA targeting GSTP1. After 72 h, GSTP1 siRNA-treated cells showed decreased GSTP1 protein expression. C: Cells, exposed to UV radiation as above, were collected and the number of Trypan blue-negative (live) cells was determined. The differential survival rate between cells unexposed and exposed to UV radiation was then calculated, and the results were expressed as percentages of dead cells after UV exposure. The results show a remarkable effect of HPV-16 E7 on cell survival (bars 1 vs. 3), a less effective protection by HPV-16 E7 Mut (bars 1 vs. 5), and the consequences of GSTP1 silencing in HPV-16 E7- (bars 3 vs. 4) and HPV-16 E7 Mut- expressing cells (bars 5 vs. 6). This panel indicates the role of GSTP1 in HPV-16 E7-induced cell survival and the importance of the binding ability of the oncoprotein in inducing the GSTP1-mediated increase in survival. Values are the average of three independent experiments performed in triplicate±SEM. Statistical significance of the reported values: bars 1 vs. 3 p = 0.00004, ***; bars 1 vs. 5 p = 0.0009, ***; bars 3 vs. 5 p = 0.000004, ***; bars 3 vs. 4 p = 0.0002, ***; bars 5 vs. 6 p = 0.0001, ***; bars 2 vs. 4 p = 0.0288, *; bars 4 vs. 6 p = 0.0007, ***.

Techniques Used: Expressing, Infection, Transfection, Binding Assay

HPV-16 E7 physically interacts with GSTP1. A: HaCaT cell lysate was incubated with the S. japonicum GST-HPV-16 E7 chimeric protein. Co-precipitated proteins were separated by SDS-PAGE and visualized after silver staining. The dotted arrow indicates the band that was cut out and identified by peptide mass fingerprinting as human GSTP1. B: In vitro interaction of radiolabeled IVT HPV-16 E7 with N-6His-GSTP1 recombinant protein and lack of interaction with control N-6His-RCC1 recombinant protein. C: Western blot for GSTP1 after immunoprecipitation with an anti-T7 Tag antibody (to precipitate tagged HPV-16 E7), in control and HPV-16 E7-transfected Phoenix cells shows co-precipitation of GSTP1 only in HPV-16 E7-expressing cells. D: Western blot for GSTP1 after immunoprecipitation with an anti-HPV-16 E7 antibody in control and HPV-16 E7-expressing HaCaT cells shows co-precipitation of GSTP1 only in HPV-16 E7-expressing cells. E: Western blot using an anti-HA antibody (to detect tagged HPV-16 E7) after immunoprecipitation with an anti-GSTP1 antibody in control and HPV-16 E7-expressing HaCaT cells shows co-precipitation of HPV-16 E7. F: Western blot for GSTP1 after immunoprecipitation with an anti-HPV-16 E7 antibody in control and HPV-16 E7-expressing HaCaT cells as well as in the CaSki and SiHa cell lines expressing endogenous HPV-16 E7. G: In vitro interaction of radiolabeled IVT HPV-33 E7 and HPV-18 E7 with N-6His-GSTP1 recombinant protein and lack of interaction with control N-6His-RCC1 recombinant protein.
Figure Legend Snippet: HPV-16 E7 physically interacts with GSTP1. A: HaCaT cell lysate was incubated with the S. japonicum GST-HPV-16 E7 chimeric protein. Co-precipitated proteins were separated by SDS-PAGE and visualized after silver staining. The dotted arrow indicates the band that was cut out and identified by peptide mass fingerprinting as human GSTP1. B: In vitro interaction of radiolabeled IVT HPV-16 E7 with N-6His-GSTP1 recombinant protein and lack of interaction with control N-6His-RCC1 recombinant protein. C: Western blot for GSTP1 after immunoprecipitation with an anti-T7 Tag antibody (to precipitate tagged HPV-16 E7), in control and HPV-16 E7-transfected Phoenix cells shows co-precipitation of GSTP1 only in HPV-16 E7-expressing cells. D: Western blot for GSTP1 after immunoprecipitation with an anti-HPV-16 E7 antibody in control and HPV-16 E7-expressing HaCaT cells shows co-precipitation of GSTP1 only in HPV-16 E7-expressing cells. E: Western blot using an anti-HA antibody (to detect tagged HPV-16 E7) after immunoprecipitation with an anti-GSTP1 antibody in control and HPV-16 E7-expressing HaCaT cells shows co-precipitation of HPV-16 E7. F: Western blot for GSTP1 after immunoprecipitation with an anti-HPV-16 E7 antibody in control and HPV-16 E7-expressing HaCaT cells as well as in the CaSki and SiHa cell lines expressing endogenous HPV-16 E7. G: In vitro interaction of radiolabeled IVT HPV-33 E7 and HPV-18 E7 with N-6His-GSTP1 recombinant protein and lack of interaction with control N-6His-RCC1 recombinant protein.

Techniques Used: Incubation, SDS Page, Silver Staining, Peptide Mass Fingerprinting, In Vitro, Recombinant, Western Blot, Immunoprecipitation, Transfection, Expressing

Docking of HPV-16 E7 to GSTP1. A: Docking of the GSTP1 monomer (from chain A of PDB entry 1AQW, represented as a protein surface) and the HPV-16 E7 CR3 dimer (modeled in this study and represented as a protein backbone with the two subunits distinguished by green and orange colors). The portions of the GSTP1 surface that contribute the Cys 47 and Cys 101 residues are highlighted in yellow. In the drawing, we have retained the structure of one glutathione molecule co-crystallized with GSTP1 in the PDB entry 1AQW to show its binding position (enzyme G-site). To show the enzymatic region that binds substrates (H-site of GSTP1), we have included the structure of the anticancer drug chlorambucil reproducing the same binding position as in its co-crystal with GSTP1 (PDB structure 21GS). B: In the docking model, the region of the GSTP1 monomer (surface representation) that is in contact with the HPV-16 E7 CR3 dimer (orange and green tube representation) is depicted in dark blue. C: The region of contact between one GSTP1 subunit (surface representation) and the other GSTP1 subunit (green tube representation) in the enzyme homodimer (PDB entry 1AQW) is depicted in dark blue. In panels B and C, regions of contact on the GSTP1 surfaces are identified as residues located within 5 Å of the ligand (HPV-16 E7 CR3 or the second GSTP1 unit) and appear to partially overlap.
Figure Legend Snippet: Docking of HPV-16 E7 to GSTP1. A: Docking of the GSTP1 monomer (from chain A of PDB entry 1AQW, represented as a protein surface) and the HPV-16 E7 CR3 dimer (modeled in this study and represented as a protein backbone with the two subunits distinguished by green and orange colors). The portions of the GSTP1 surface that contribute the Cys 47 and Cys 101 residues are highlighted in yellow. In the drawing, we have retained the structure of one glutathione molecule co-crystallized with GSTP1 in the PDB entry 1AQW to show its binding position (enzyme G-site). To show the enzymatic region that binds substrates (H-site of GSTP1), we have included the structure of the anticancer drug chlorambucil reproducing the same binding position as in its co-crystal with GSTP1 (PDB structure 21GS). B: In the docking model, the region of the GSTP1 monomer (surface representation) that is in contact with the HPV-16 E7 CR3 dimer (orange and green tube representation) is depicted in dark blue. C: The region of contact between one GSTP1 subunit (surface representation) and the other GSTP1 subunit (green tube representation) in the enzyme homodimer (PDB entry 1AQW) is depicted in dark blue. In panels B and C, regions of contact on the GSTP1 surfaces are identified as residues located within 5 Å of the ligand (HPV-16 E7 CR3 or the second GSTP1 unit) and appear to partially overlap.

Techniques Used: Binding Assay

Identification of HPV-16 E7 peptide sequences involved in the interaction with GSTP1. The entire HPV-16 E7 amino acid sequence, reproduced as 12-mers with a 9 amino acid overlap, was synthesized on 30 different polypropylene rods. The histogram represents the ability of each 12-mer oligopeptide to bind recombinant GSTP1. The histogram corresponding to the 30 th rod refers to amino acids 88–99 because a Gly has been added to the last C-terminal HPV-16 E7 amino acid in order to complete the 12-mer. Values are averages of three different experiments±SD.
Figure Legend Snippet: Identification of HPV-16 E7 peptide sequences involved in the interaction with GSTP1. The entire HPV-16 E7 amino acid sequence, reproduced as 12-mers with a 9 amino acid overlap, was synthesized on 30 different polypropylene rods. The histogram represents the ability of each 12-mer oligopeptide to bind recombinant GSTP1. The histogram corresponding to the 30 th rod refers to amino acids 88–99 because a Gly has been added to the last C-terminal HPV-16 E7 amino acid in order to complete the 12-mer. Values are averages of three different experiments±SD.

Techniques Used: Sequencing, Synthesized, Recombinant

GSTP1 activities in HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells. A: A representative Western blot for GSTP1 in control cells and HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells. Blots were normalized against β-actin levels. B: GSTP1 enzymatic activity in control, in HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells (see Materials and Methods ). Values are averages of three different experiments±SEM and their variations were not statistically significant. C: JNK protein levels in control and in HPV-16 E7- and in HPV-16 E7 Mut-expressing HaCaT cells that were untreated or cells that were sampled after induction of oxidative stress by exposure to UV (UVB, 5 mJ/cm 2 for 1 min) with a 5 h recovery period, evaluated using an antibody specific for the phosphorylated form of JNK (pJNK) and another antibody that detects total JNK levels (JNK, performed on a separate twin gel). Only after UV exposure did HPV-16 E7-expressing cells display a markedly reduced JNK phosphorylation, while a lower reduction in JNK phosphorylation was detectable in HPV-16 E7 Mut-expressing cells.
Figure Legend Snippet: GSTP1 activities in HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells. A: A representative Western blot for GSTP1 in control cells and HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells. Blots were normalized against β-actin levels. B: GSTP1 enzymatic activity in control, in HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells (see Materials and Methods ). Values are averages of three different experiments±SEM and their variations were not statistically significant. C: JNK protein levels in control and in HPV-16 E7- and in HPV-16 E7 Mut-expressing HaCaT cells that were untreated or cells that were sampled after induction of oxidative stress by exposure to UV (UVB, 5 mJ/cm 2 for 1 min) with a 5 h recovery period, evaluated using an antibody specific for the phosphorylated form of JNK (pJNK) and another antibody that detects total JNK levels (JNK, performed on a separate twin gel). Only after UV exposure did HPV-16 E7-expressing cells display a markedly reduced JNK phosphorylation, while a lower reduction in JNK phosphorylation was detectable in HPV-16 E7 Mut-expressing cells.

Techniques Used: Expressing, Western Blot, Activity Assay

Generation of HPV-16 E7 Mut and its in vitro interaction with GSTP1 and pRb. A. Legend as in Figure 3A . The red spheres shown on one HPV-16 E7 subunit (green tube representation) highlight the alpha-carbon atoms of amino acid residues 49–60, while the blue spheres highlight the alpha-carbons of amino acid residues 88–98 (see Figure 2 ). The residues Val 55, Phe 57 and Met 84, mutated in HPV-16 E7 Mut, are highlighted by the white clouds. B: In vitro interaction of radiolabeled IVT HPV-16 E7, but not of HPV-16 E7 Mut, with N-6His-GSTP1 recombinant protein. N-6His-RCC1 recombinant protein was used as a negative control. C: In vitro interaction of radiolabeled IVT HPV-16 E7 and HPV-16 E7 Mut with recombinant S. japonicum GST-pRb protein. S. japonicum GST was used as a negative control. D: Western blot for GSTP1 after immunoprecipitation with an anti-HA antibody (to precipitate tagged HPV-16 E7 and HPV-16 E7 Mut), in control, HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells shows a less efficient co-precipitation of GSTP1 in HPV-16 E7 Mut-expressing cells; the same membrane was probed using an anti-HA antibody to assess the efficiency of the immunoprecipitation procedure. Cell lysate was from HPV-16 E7-infected HaCaT cells. E: Reverse co-precipitation: Western blot for HA (to detect tagged HPV-16 E7 and HPV-16 E7 Mut) after immunoprecipitation with an anti-GSTP1 antibody in control, HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells shows a less efficient co-precipitation of HPV-16 E7 Mut with GSTP1 when compared to HPV-16 E7; the same membrane was probed using an anti-GSTP1 antibody to assess the efficiency of the immunoprecipitation procedure. Cell lysate was from HPV-16 E7-infected HaCaT cells.
Figure Legend Snippet: Generation of HPV-16 E7 Mut and its in vitro interaction with GSTP1 and pRb. A. Legend as in Figure 3A . The red spheres shown on one HPV-16 E7 subunit (green tube representation) highlight the alpha-carbon atoms of amino acid residues 49–60, while the blue spheres highlight the alpha-carbons of amino acid residues 88–98 (see Figure 2 ). The residues Val 55, Phe 57 and Met 84, mutated in HPV-16 E7 Mut, are highlighted by the white clouds. B: In vitro interaction of radiolabeled IVT HPV-16 E7, but not of HPV-16 E7 Mut, with N-6His-GSTP1 recombinant protein. N-6His-RCC1 recombinant protein was used as a negative control. C: In vitro interaction of radiolabeled IVT HPV-16 E7 and HPV-16 E7 Mut with recombinant S. japonicum GST-pRb protein. S. japonicum GST was used as a negative control. D: Western blot for GSTP1 after immunoprecipitation with an anti-HA antibody (to precipitate tagged HPV-16 E7 and HPV-16 E7 Mut), in control, HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells shows a less efficient co-precipitation of GSTP1 in HPV-16 E7 Mut-expressing cells; the same membrane was probed using an anti-HA antibody to assess the efficiency of the immunoprecipitation procedure. Cell lysate was from HPV-16 E7-infected HaCaT cells. E: Reverse co-precipitation: Western blot for HA (to detect tagged HPV-16 E7 and HPV-16 E7 Mut) after immunoprecipitation with an anti-GSTP1 antibody in control, HPV-16 E7- and HPV-16 E7 Mut-expressing HaCaT cells shows a less efficient co-precipitation of HPV-16 E7 Mut with GSTP1 when compared to HPV-16 E7; the same membrane was probed using an anti-GSTP1 antibody to assess the efficiency of the immunoprecipitation procedure. Cell lysate was from HPV-16 E7-infected HaCaT cells.

Techniques Used: In Vitro, Recombinant, Negative Control, Western Blot, Immunoprecipitation, Expressing, Infection

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

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

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M806152200

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

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

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

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

27) Product Images from "The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell's Response to LPA"

Article Title: The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell's Response to LPA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049227

GIPC directly interacts with the C-terminal PDZ binding motif of LPA 1 but not with other LPA receptors. A, Endogenous GIPC and GIPC-GFP co-immunoprecipitate with FLAG- LPA 1 from HEK cells expressing FLAG- LPA 1 (arrowhead, lane 4) but not control HEK cells (lane 3). C-terminally tagged GIPC-GFP and N-terminally tagged FLAG-LPA 1 were transiently coexpressed in HEK293 cells, and immunoprecipitation was carried out on cell lysates with mouse anti-FLAG IgG followed by immunoblotting with mouse anti-FLAG (LPA 1 ) and rabbit anti-GIPC IgG. Lanes were cropped from a single exposure of a continuous membrane. The lower panel shows the amount of IgG light-chain (IgG-LC) in each IP. Lanes 1–2 : Input showing the amounts of LPA 1 and GIPC present in the lysates used for the IP. B, Upper panel : GIPC binds GST-LPA 1 (GST fused to the cytoplasmic tail of mouse LPA 1 (aa 311–364), lane 3) but not to GST alone (lane 2) or GST-LPA 1 ΔC (lacking the last three C-terminal amino acids, lane 4). Immobilized recombinant GST, GST-LPA 1 and GST- LPA 1 ΔC were incubated 4–15 h with lysates from HEK293 cells transiently transfected with FLAG-GIPC. Proteins bound to immobilized fusion proteins were eluted with 2X sample buffer for SDS-PAGE and immunoblotted with anti-GIPC IgG. Lane 1: input, showing the amount of GIPC in 1% of the lysate used for the assay. Lower panel : Ponceau staining demonstrating the amount of GST proteins used in each assay. C, Upper panel : Autoradiography showing that in vitro translated, [ 35 S]GIPC PDZ domain binds to GST-LPA 1 (lane 3) but not to GST alone (lane 2), GST- LPA 1 AAA (last three amino acids mutated to alanine, lane 4), or GST-LPA 2 (lane 5). GST fusion proteins were immobilized on glutathione-agarose beads as in “B” and incubated with in vitro translated [ 35 S]Met-labeled, GIPC PDZ domain (aa 125–225). Bound proteins were separated by SDS-PAGE and detected by autoradiography. Lane 1: 5% of the in vitro translated protein. Lower panel : Coomassie Blue staining showing the GST proteins used for the assay. D, Upper panel: Autoradiography showing that in vitro translated, [ 35 S] GIPC-PDZ interacts with the C-terminal PDZ binding motif of LPA 1 (SVV, lane 3) and with GST-GAIP (lane 8, used as a positive control [7] but shows little or no interaction with GST alone (lane 2) or GST-LPA 1 mutants in which the three C-terminal amino acids were modified to those of LPA 2 (STL, lane 4), LPA 3 (NGS, lane 5), LPA 4 (STF, lane 6) or LPA 5 (SAL, lane 7). Immobilized GST fusion proteins were incubated with in vitro translated [ 35 S]Met-labeled GIPC PDZ and analyzed as in C. Lower panel : Coomassie Blue staining showing the amounts of GST proteins used.
Figure Legend Snippet: GIPC directly interacts with the C-terminal PDZ binding motif of LPA 1 but not with other LPA receptors. A, Endogenous GIPC and GIPC-GFP co-immunoprecipitate with FLAG- LPA 1 from HEK cells expressing FLAG- LPA 1 (arrowhead, lane 4) but not control HEK cells (lane 3). C-terminally tagged GIPC-GFP and N-terminally tagged FLAG-LPA 1 were transiently coexpressed in HEK293 cells, and immunoprecipitation was carried out on cell lysates with mouse anti-FLAG IgG followed by immunoblotting with mouse anti-FLAG (LPA 1 ) and rabbit anti-GIPC IgG. Lanes were cropped from a single exposure of a continuous membrane. The lower panel shows the amount of IgG light-chain (IgG-LC) in each IP. Lanes 1–2 : Input showing the amounts of LPA 1 and GIPC present in the lysates used for the IP. B, Upper panel : GIPC binds GST-LPA 1 (GST fused to the cytoplasmic tail of mouse LPA 1 (aa 311–364), lane 3) but not to GST alone (lane 2) or GST-LPA 1 ΔC (lacking the last three C-terminal amino acids, lane 4). Immobilized recombinant GST, GST-LPA 1 and GST- LPA 1 ΔC were incubated 4–15 h with lysates from HEK293 cells transiently transfected with FLAG-GIPC. Proteins bound to immobilized fusion proteins were eluted with 2X sample buffer for SDS-PAGE and immunoblotted with anti-GIPC IgG. Lane 1: input, showing the amount of GIPC in 1% of the lysate used for the assay. Lower panel : Ponceau staining demonstrating the amount of GST proteins used in each assay. C, Upper panel : Autoradiography showing that in vitro translated, [ 35 S]GIPC PDZ domain binds to GST-LPA 1 (lane 3) but not to GST alone (lane 2), GST- LPA 1 AAA (last three amino acids mutated to alanine, lane 4), or GST-LPA 2 (lane 5). GST fusion proteins were immobilized on glutathione-agarose beads as in “B” and incubated with in vitro translated [ 35 S]Met-labeled, GIPC PDZ domain (aa 125–225). Bound proteins were separated by SDS-PAGE and detected by autoradiography. Lane 1: 5% of the in vitro translated protein. Lower panel : Coomassie Blue staining showing the GST proteins used for the assay. D, Upper panel: Autoradiography showing that in vitro translated, [ 35 S] GIPC-PDZ interacts with the C-terminal PDZ binding motif of LPA 1 (SVV, lane 3) and with GST-GAIP (lane 8, used as a positive control [7] but shows little or no interaction with GST alone (lane 2) or GST-LPA 1 mutants in which the three C-terminal amino acids were modified to those of LPA 2 (STL, lane 4), LPA 3 (NGS, lane 5), LPA 4 (STF, lane 6) or LPA 5 (SAL, lane 7). Immobilized GST fusion proteins were incubated with in vitro translated [ 35 S]Met-labeled GIPC PDZ and analyzed as in C. Lower panel : Coomassie Blue staining showing the amounts of GST proteins used.

Techniques Used: Binding Assay, Expressing, Immunoprecipitation, Recombinant, Incubation, Transfection, SDS Page, Staining, Autoradiography, In Vitro, Labeling, Positive Control, Modification, Next-Generation Sequencing

28) Product Images from "Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus"

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054075

Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).
Figure Legend Snippet: Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).

Techniques Used: Transfection

Interaction between Rta and TAF4 in vitro . GST-TAF4 (lanes 3 and 6) or GST (lanes 2, 5) was added to the lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate (lanes 1–3) or from E. coli BL21(DE3)(pET-Rta) (lanes 4–6). Proteins bound to GST-TAF4 were pulled down by glutathione-Sepharose beads and analyzed by immunoblotting (IB) with anti-Rta antibody. Lanes 1 and 4 were loaded with 5% of cell lysate. GST or GST-TAF4 bound to glutathione-Sepharose beads were analyzed by immunoblotting (IB) with anti-GST antibody (lanes 7, 8).
Figure Legend Snippet: Interaction between Rta and TAF4 in vitro . GST-TAF4 (lanes 3 and 6) or GST (lanes 2, 5) was added to the lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate (lanes 1–3) or from E. coli BL21(DE3)(pET-Rta) (lanes 4–6). Proteins bound to GST-TAF4 were pulled down by glutathione-Sepharose beads and analyzed by immunoblotting (IB) with anti-Rta antibody. Lanes 1 and 4 were loaded with 5% of cell lysate. GST or GST-TAF4 bound to glutathione-Sepharose beads were analyzed by immunoblotting (IB) with anti-GST antibody (lanes 7, 8).

Techniques Used: In Vitro

Indirect immunofluorescence analysis. P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.
Figure Legend Snippet: Indirect immunofluorescence analysis. P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.

Techniques Used: Immunofluorescence, Transfection, Incubation, Laser-Scanning Microscopy, Staining

Influence of TAF4 on the activation of EBV TATA-less promoters by Rta. (A) The TR-L1 reporter plasmid and sequences of biotin-labeled probes, TR-L1 and mTR-L1. The probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate. Proteins bound to the probes were captured by streptavidin magnetic beads and detected by immunoblotting analysis using anti-Sp1, anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA-affinity precipitation assay. (B) P3HR1 cells that had been treated with TPA and sodium butyrate for 48 hr (lytic) or treated with DMSO (latent) were fixed with formaldehyde and the DNA-protein complexes were immunoprecipitated using anti-Sp1, anti-TAF4 and anti-Rta antibodies. The binding of Sp1, TAF4 and Rta to the TR-L1 and BALF5 promoters was investigated by qPCR. Error bar represents standard error. (C) 293T cells were cotransfected with pCMV-R and reporter plasmids, including pTRL1-luc and pBALF5-luc in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (empty column). Luciferase activities were monitored at 48 hr after transfection. Each transfection experiment was performed at lease three times, and each sample in the experiment was prepared in duplicate. (D) A similar experiment in (C) was performed using pBMRF1-luc and pBMRF1-mRRE-luc. (E) The effect of TAF4 shRNAs on the expression of TAF4 was examined by immunoblotting using anti-TAF4 and anti-α-tubulin antibodies. The p value from each experiment was analyzed statistically with the Student's t- test method. Luc: luciferase gene.
Figure Legend Snippet: Influence of TAF4 on the activation of EBV TATA-less promoters by Rta. (A) The TR-L1 reporter plasmid and sequences of biotin-labeled probes, TR-L1 and mTR-L1. The probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate. Proteins bound to the probes were captured by streptavidin magnetic beads and detected by immunoblotting analysis using anti-Sp1, anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA-affinity precipitation assay. (B) P3HR1 cells that had been treated with TPA and sodium butyrate for 48 hr (lytic) or treated with DMSO (latent) were fixed with formaldehyde and the DNA-protein complexes were immunoprecipitated using anti-Sp1, anti-TAF4 and anti-Rta antibodies. The binding of Sp1, TAF4 and Rta to the TR-L1 and BALF5 promoters was investigated by qPCR. Error bar represents standard error. (C) 293T cells were cotransfected with pCMV-R and reporter plasmids, including pTRL1-luc and pBALF5-luc in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (empty column). Luciferase activities were monitored at 48 hr after transfection. Each transfection experiment was performed at lease three times, and each sample in the experiment was prepared in duplicate. (D) A similar experiment in (C) was performed using pBMRF1-luc and pBMRF1-mRRE-luc. (E) The effect of TAF4 shRNAs on the expression of TAF4 was examined by immunoblotting using anti-TAF4 and anti-α-tubulin antibodies. The p value from each experiment was analyzed statistically with the Student's t- test method. Luc: luciferase gene.

Techniques Used: Activation Assay, Plasmid Preparation, Labeling, Magnetic Beads, Affinity Precipitation, Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, shRNA, Luciferase, Transfection, Expressing

Role of TAF4 in the transcription of human androgen receptor by Rta. (A) An hAR reporter plasmid and sequences of biotin-labeled probes, hAR and mhAR. The probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate. Proteins bound to the probes were captured by streptavidin magnetic beads and detected by immunoblot analysis using anti-Sp1, anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA-affinity precipitation assay. (B) P3HR1 cells that had been treated with TPA and sodium butyrate for 48 hr (lytic) or treated with DMSO (latent) were fixed with formaldehyde and the DNA-protein complexes were immunoprecipitated with anti-Sp1, anti-TAF4 and anti-Rta antibodies. The binding of Sp1, TAF4 and Rta to the hAR promoter was investigated by qPCR. (C) 293T cells were cotransfected with pCMV-R and TAF4 shRNA (empty column) or control shRNA (filled column) for 48 hr. CHIP assay was subsequently performed. The reaction with added anti-IgG antibody was used as a negative control. Error bar represents standard error. (D) 293T cells were cotransfected with pCMV-R and a reporter plasmid including phAR-luc or pmhAR-luc, in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (empty column). Luciferase activities were monitored at 48 hr after transfection. Each transfection experiment was performed at lease three times, and each sample in the experiment was prepared in duplicate. The p value from each experiment was analyzed statistically with the Student's t- test method. Luc: luciferase gene.
Figure Legend Snippet: Role of TAF4 in the transcription of human androgen receptor by Rta. (A) An hAR reporter plasmid and sequences of biotin-labeled probes, hAR and mhAR. The probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate. Proteins bound to the probes were captured by streptavidin magnetic beads and detected by immunoblot analysis using anti-Sp1, anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA-affinity precipitation assay. (B) P3HR1 cells that had been treated with TPA and sodium butyrate for 48 hr (lytic) or treated with DMSO (latent) were fixed with formaldehyde and the DNA-protein complexes were immunoprecipitated with anti-Sp1, anti-TAF4 and anti-Rta antibodies. The binding of Sp1, TAF4 and Rta to the hAR promoter was investigated by qPCR. (C) 293T cells were cotransfected with pCMV-R and TAF4 shRNA (empty column) or control shRNA (filled column) for 48 hr. CHIP assay was subsequently performed. The reaction with added anti-IgG antibody was used as a negative control. Error bar represents standard error. (D) 293T cells were cotransfected with pCMV-R and a reporter plasmid including phAR-luc or pmhAR-luc, in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (empty column). Luciferase activities were monitored at 48 hr after transfection. Each transfection experiment was performed at lease three times, and each sample in the experiment was prepared in duplicate. The p value from each experiment was analyzed statistically with the Student's t- test method. Luc: luciferase gene.

Techniques Used: Plasmid Preparation, Labeling, Magnetic Beads, Affinity Precipitation, Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, shRNA, Chromatin Immunoprecipitation, Negative Control, Luciferase, Transfection

Involvement of TAF4 in the activation of the BcLF1 promoter by Rta. (A) Biotin-labeled double-stranded F23 probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate for 52 hr. Mutant probes mF23 that contains mutated sequences (asterisk) was used as negative controls. Proteins bound to the probes were captured by the streptavidin magnetic beads and detected by immunoblotting analysis using anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA affinity precipitation assay. (B) P3HR1 cells were treated with TPA and sodium butyrate for 52 hr. Formaldehyde-fixed DNA-protein complex was immunoprecipitated using anti-TAF4 or anti-Rta antibody. The reaction with added anti-IgG antibody was used as a negative control. The binding of TAF4 and Rta to the BcLF1 promoter was investigated by qPCR. Error bar represents standard error. (C) 293T cells were cotransfected with pCMV-R and pGL2-F23 or a control vector pGL2-basic in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (shTAF4) (empty column). Luciferase activity was detected at 48 hr after transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate. (D) The effect of TAF4 shRNAs was examined by immunoblotting with anti-TAF4 and anti-α-tubulin antibodies. The p value from each experiment was analyzed statistically with the Student's t- test method.
Figure Legend Snippet: Involvement of TAF4 in the activation of the BcLF1 promoter by Rta. (A) Biotin-labeled double-stranded F23 probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate for 52 hr. Mutant probes mF23 that contains mutated sequences (asterisk) was used as negative controls. Proteins bound to the probes were captured by the streptavidin magnetic beads and detected by immunoblotting analysis using anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA affinity precipitation assay. (B) P3HR1 cells were treated with TPA and sodium butyrate for 52 hr. Formaldehyde-fixed DNA-protein complex was immunoprecipitated using anti-TAF4 or anti-Rta antibody. The reaction with added anti-IgG antibody was used as a negative control. The binding of TAF4 and Rta to the BcLF1 promoter was investigated by qPCR. Error bar represents standard error. (C) 293T cells were cotransfected with pCMV-R and pGL2-F23 or a control vector pGL2-basic in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (shTAF4) (empty column). Luciferase activity was detected at 48 hr after transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate. (D) The effect of TAF4 shRNAs was examined by immunoblotting with anti-TAF4 and anti-α-tubulin antibodies. The p value from each experiment was analyzed statistically with the Student's t- test method.

Techniques Used: Activation Assay, Labeling, Mutagenesis, Magnetic Beads, Affinity Precipitation, Immunoprecipitation, Negative Control, Binding Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, shRNA, Luciferase, Activity Assay, Transfection

Coimmunoprecipitation of Rta and TAF4. Interaction between TAF4 and Rta was examined using P3HR1 cells that were treated with DMSO (lanes 5–8; 13–16) or TPA and sodium butyrate (lanes 1–4; 9–12) for 24 hr. Proteins in the cell lysate were immunoprecipitated with anti-Rta (lanes 4, 8, 11, 15), anti-TAF4 (lanes 3, 7, 12, 16) or anti-IgG (lanes 2, 6, 10, 14) antibody. Immunoprecipitated proteins were detected by immunoblotting with anti-Rta (lanes 1–8) or anti-TAF4 (lanes 9–16) antibody. Lanes 1, 5, 9 and 13 were loaded with 5% of the cell lysate.
Figure Legend Snippet: Coimmunoprecipitation of Rta and TAF4. Interaction between TAF4 and Rta was examined using P3HR1 cells that were treated with DMSO (lanes 5–8; 13–16) or TPA and sodium butyrate (lanes 1–4; 9–12) for 24 hr. Proteins in the cell lysate were immunoprecipitated with anti-Rta (lanes 4, 8, 11, 15), anti-TAF4 (lanes 3, 7, 12, 16) or anti-IgG (lanes 2, 6, 10, 14) antibody. Immunoprecipitated proteins were detected by immunoblotting with anti-Rta (lanes 1–8) or anti-TAF4 (lanes 9–16) antibody. Lanes 1, 5, 9 and 13 were loaded with 5% of the cell lysate.

Techniques Used: Immunoprecipitation

29) Product Images from "Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function"

Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

Journal: Retrovirology

doi: 10.1186/1742-4690-3-83

Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a glutathione-Sepharose 4B column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).
Figure Legend Snippet: Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a glutathione-Sepharose 4B column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).

Techniques Used: Affinity Chromatography, Recombinant, Transfection, Purification

30) Product Images from "Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking"

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

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15022929

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.
Figure Legend 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.

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

31) Product Images from "The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex"

Article Title: The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00270-13

The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of
Figure Legend Snippet: The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of

Techniques Used: Recombinant, Incubation

32) Product Images from "CHLOROPLAST UNUSUAL POSITIONING 1 is a new type of actin nucleation factor in plants"

Article Title: CHLOROPLAST UNUSUAL POSITIONING 1 is a new type of actin nucleation factor in plants

Journal: bioRxiv

doi: 10.1101/2020.01.14.905984

Effects of mutations at R820 and F958 of CHUP1-C on the activity of CHUP1 (A) A schematic illustration of CHUP1 protein showing the functional domains: the hydrophobic region, the coiled-coil domain, the F-actin binding domain, the proline-rich FH1-like domain, and the FH2-like domain. Asterisks indicate R820, which is involved in the association with actin, and F958, which is involved in the dimerization of the FH2-like domain. The positions of the 611–1004 fragment (CHUP1-C) and the 611–982 and 716–982 deletion mutations are indicated by lines below the structure. (B–C) The effect of point mutations R820D and F958D on CHUP1-C dimer structure. (B) The elution profiles of CHUP1-C_WT, R820D, and F958D recombinant proteins were obtained by gel filtration chromatography using a Superdex 200 10/300 column. (C) The protein profiles were confirmed using 10% SDS-PAGE gels. (D–E) Ultracentrifugation assay of actin polymerization. ACT7 (4 µM) was allowed to polymerize in F-buffer containing 8 µM PRF1 and 0.4 µM WT or mutant CHUP1-C. (D) A representative SDS-PAGE gel of supernatant (S) and pellet (P) fractions after ultracentrifugation. (E) A bar graph showing the fraction of ACT7 in the pellet and the supernatant under each condition, with error bars showing SD (N = 3). (F) Immunoblot analysis showing the expression levels of CHUP1-YFP_R820D and CHUP1-YFP_F985D in C1Y_R820D (18-2 and 26-3) and C1Y_F958D (6-5 and 10-1) transgenic plants. The arrowhead and arrow indicate CHUP1 and CHUP1-Y, respectively. * indicates truncated CHUP1 and CHUP1-Y. The details are the same as in   Figure 2A . (G) Chloroplast photorelocation movement in WT,  chup1  mutant ( chup1-3 ), and C1Y_R820D and C1Y_F985D transgenic plants in a  chup1  background. The red light (RL) transmittance in rosette leaves was monitored under different intensities of blue light (BL; 0, 2.8, and 50 µmol m −2  s −1 ) for the indicated periods.
Figure Legend Snippet: Effects of mutations at R820 and F958 of CHUP1-C on the activity of CHUP1 (A) A schematic illustration of CHUP1 protein showing the functional domains: the hydrophobic region, the coiled-coil domain, the F-actin binding domain, the proline-rich FH1-like domain, and the FH2-like domain. Asterisks indicate R820, which is involved in the association with actin, and F958, which is involved in the dimerization of the FH2-like domain. The positions of the 611–1004 fragment (CHUP1-C) and the 611–982 and 716–982 deletion mutations are indicated by lines below the structure. (B–C) The effect of point mutations R820D and F958D on CHUP1-C dimer structure. (B) The elution profiles of CHUP1-C_WT, R820D, and F958D recombinant proteins were obtained by gel filtration chromatography using a Superdex 200 10/300 column. (C) The protein profiles were confirmed using 10% SDS-PAGE gels. (D–E) Ultracentrifugation assay of actin polymerization. ACT7 (4 µM) was allowed to polymerize in F-buffer containing 8 µM PRF1 and 0.4 µM WT or mutant CHUP1-C. (D) A representative SDS-PAGE gel of supernatant (S) and pellet (P) fractions after ultracentrifugation. (E) A bar graph showing the fraction of ACT7 in the pellet and the supernatant under each condition, with error bars showing SD (N = 3). (F) Immunoblot analysis showing the expression levels of CHUP1-YFP_R820D and CHUP1-YFP_F985D in C1Y_R820D (18-2 and 26-3) and C1Y_F958D (6-5 and 10-1) transgenic plants. The arrowhead and arrow indicate CHUP1 and CHUP1-Y, respectively. * indicates truncated CHUP1 and CHUP1-Y. The details are the same as in Figure 2A . (G) Chloroplast photorelocation movement in WT, chup1 mutant ( chup1-3 ), and C1Y_R820D and C1Y_F985D transgenic plants in a chup1 background. The red light (RL) transmittance in rosette leaves was monitored under different intensities of blue light (BL; 0, 2.8, and 50 µmol m −2 s −1 ) for the indicated periods.

Techniques Used: Activity Assay, Functional Assay, Binding Assay, Recombinant, Filtration, Chromatography, SDS Page, Mutagenesis, Expressing, Transgenic Assay

33) Product Images from "Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores"

Article Title: Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201407074

CENP-T contributes to KMN kinetochore targeting independently of its NBM. (A) HeLa cells were mock transfected or transfected with the indicated siRNAs, treated with thymidine for 14 h, released into nocodazole-containing medium for 12 h, and treated with ZM for 2 h. The mitotic indices of these cells (mean ± SD [error bars], n = 3) were quantified with FACS. (B) Lysates of cells in A were blotted with the indicated antibodies. (C) HeLa cells were mock transfected or transfected with siCENP-T and arrested in mitosis by nocodazole. Mitotic cells were collected by shake-off. Each sample was divided into two fresh wells. One well was incubated with MG132 (MG) for 2 h (NM) while the other well was treated with both MG and ZM for 2 h (NM+Z). Cells were stained with the indicated antibodies and DAPI. The boxed regions of the merged images of the selected channels were magnified and shown in the rightmost column. The relative kinetochore intensities (mean ± SD, n = 400) of CENP-C and Mis12C were quantified and shown. Bars, 5 µm (1 µm for magnified images). (D) Lysates (Input), α-Mis12C IP (Mis12C), and IgG IP of mitotic HeLa cells transfected with the indicated siRNAs and treated with nocodazole were blotted with the indicated antibodies. (E) In vitro binding assay between recombinant Mis12C and Spc24-25 in the presence of increasing concentrations of a synthetic peptide containing residues 80–104 of human CENP-T with phospho-T85. The reaction mixtures were blotted with anti-Mis12C and anti-Spc24/25 antibodies. (F) Lysates (Input) and α-Mis12C IP (Mis12C) of mitotic HeLa cells transfected with the indicated plasmids and siCENP-C and treated with nocodazole were blotted with the indicated antibodies. Residues 85–99 were deleted in CENP-T ΔNBM. Lines indicate that intervening lanes have been spliced out. (G) Quantification of mitotic indices of HeLa cells expressing indicated proteins that were transfected with siCENP-T and treated with nocodazole and ZM (mean ± SD [error bars], n = 3).
Figure Legend Snippet: CENP-T contributes to KMN kinetochore targeting independently of its NBM. (A) HeLa cells were mock transfected or transfected with the indicated siRNAs, treated with thymidine for 14 h, released into nocodazole-containing medium for 12 h, and treated with ZM for 2 h. The mitotic indices of these cells (mean ± SD [error bars], n = 3) were quantified with FACS. (B) Lysates of cells in A were blotted with the indicated antibodies. (C) HeLa cells were mock transfected or transfected with siCENP-T and arrested in mitosis by nocodazole. Mitotic cells were collected by shake-off. Each sample was divided into two fresh wells. One well was incubated with MG132 (MG) for 2 h (NM) while the other well was treated with both MG and ZM for 2 h (NM+Z). Cells were stained with the indicated antibodies and DAPI. The boxed regions of the merged images of the selected channels were magnified and shown in the rightmost column. The relative kinetochore intensities (mean ± SD, n = 400) of CENP-C and Mis12C were quantified and shown. Bars, 5 µm (1 µm for magnified images). (D) Lysates (Input), α-Mis12C IP (Mis12C), and IgG IP of mitotic HeLa cells transfected with the indicated siRNAs and treated with nocodazole were blotted with the indicated antibodies. (E) In vitro binding assay between recombinant Mis12C and Spc24-25 in the presence of increasing concentrations of a synthetic peptide containing residues 80–104 of human CENP-T with phospho-T85. The reaction mixtures were blotted with anti-Mis12C and anti-Spc24/25 antibodies. (F) Lysates (Input) and α-Mis12C IP (Mis12C) of mitotic HeLa cells transfected with the indicated plasmids and siCENP-C and treated with nocodazole were blotted with the indicated antibodies. Residues 85–99 were deleted in CENP-T ΔNBM. Lines indicate that intervening lanes have been spliced out. (G) Quantification of mitotic indices of HeLa cells expressing indicated proteins that were transfected with siCENP-T and treated with nocodazole and ZM (mean ± SD [error bars], n = 3).

Techniques Used: Transfection, FACS, Incubation, Staining, In Vitro, Binding Assay, Recombinant, Expressing

Phospho-mimicking Dsn1 mutation enhances Mis12C binding to CENP-C. (A) HeLa cells expressing Dsn1-WT/EE-GFP were analyzed by time-lapse microscopy. GFP images of representative cells at the indicated times (in minutes) were shown. Metaphase was used as the reference point (time 0). (B) Interphase cells expressing Dsn1-WT/EE-GFP were stained with the indicated antibodies and DAPI. (C–E) HeLa cells expressing Dsn1-WT/EE-GFP were transfected with Ndc80-WT/NLS-mCherry plasmids, and stained with the indicated antibodies and DAPI. (F) HeLa cells expressing Dsn1-EE-GFP transfected with siCENP-C and treated with thymidine were stained with the indicated antibodies and DAPI. (G) In vitro binding assays between recombinant Dsn1-WT/EE-containing Mis12C with or without Ndc80C and 35 S-labeled CENP-C (residues 1–71; right). The relative CENP-C binding intensity is indicated at the bottom. Mis12C and Ndc80C were stained with CBB (left). The asterisk indicates a degradation band of Ndc80. (H) Sequence alignment of the basic motif encompassing the two Aurora B phosphorylation sites in Dsn1 proteins from different species (Hs, Homo sapiens ; Mm, Mus musculus ; Gg, Gallus gallus ; Xl, Xenopus laevis ). (I) HeLa cells expressing Dsn1-WT/Δ92-113-GFP were analyzed by time-lapse microscopy. (J) Lysates and α-GFP IP of mitotic HeLa cells expressing Dsn1-WT, -AA, -EE, or -Δ92–113-GFP were blotted with the indicated antibodies. Bars: (A and I) 10 µm; (B–F) 5 µm.
Figure Legend Snippet: Phospho-mimicking Dsn1 mutation enhances Mis12C binding to CENP-C. (A) HeLa cells expressing Dsn1-WT/EE-GFP were analyzed by time-lapse microscopy. GFP images of representative cells at the indicated times (in minutes) were shown. Metaphase was used as the reference point (time 0). (B) Interphase cells expressing Dsn1-WT/EE-GFP were stained with the indicated antibodies and DAPI. (C–E) HeLa cells expressing Dsn1-WT/EE-GFP were transfected with Ndc80-WT/NLS-mCherry plasmids, and stained with the indicated antibodies and DAPI. (F) HeLa cells expressing Dsn1-EE-GFP transfected with siCENP-C and treated with thymidine were stained with the indicated antibodies and DAPI. (G) In vitro binding assays between recombinant Dsn1-WT/EE-containing Mis12C with or without Ndc80C and 35 S-labeled CENP-C (residues 1–71; right). The relative CENP-C binding intensity is indicated at the bottom. Mis12C and Ndc80C were stained with CBB (left). The asterisk indicates a degradation band of Ndc80. (H) Sequence alignment of the basic motif encompassing the two Aurora B phosphorylation sites in Dsn1 proteins from different species (Hs, Homo sapiens ; Mm, Mus musculus ; Gg, Gallus gallus ; Xl, Xenopus laevis ). (I) HeLa cells expressing Dsn1-WT/Δ92-113-GFP were analyzed by time-lapse microscopy. (J) Lysates and α-GFP IP of mitotic HeLa cells expressing Dsn1-WT, -AA, -EE, or -Δ92–113-GFP were blotted with the indicated antibodies. Bars: (A and I) 10 µm; (B–F) 5 µm.

Techniques Used: Mutagenesis, Binding Assay, Expressing, Time-lapse Microscopy, Staining, Transfection, In Vitro, Recombinant, Labeling, Sequencing

34) Product Images from "Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA"

Article Title: Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00341-15

Screen for Bicc1 SAM polymer mutants. (A) Bicc1 SAM mutant collection. Shading is as for Fig, 3A . Individual electrostatic patches (patches A to F) at the protein surface were replaced by alanines. (B, C) Positions of mutations in the Bicc1 SAM dimer model (B) and on the ML surface and EH surface (C). (D) Table summarizing, for each amino acid patch, the average number of H bonds per time frame during the MD. (E and F) Pulldown of the WT, point mutants, or 8-fold excess bpk mutant HA-tagged Bicc1 from HEK293T cell extracts by glutathione-Sepharose beads coated with a recombinant GST control or GST-Bicc1 SAM. Five percent of total cell extracts were loaded as input.
Figure Legend Snippet: Screen for Bicc1 SAM polymer mutants. (A) Bicc1 SAM mutant collection. Shading is as for Fig, 3A . Individual electrostatic patches (patches A to F) at the protein surface were replaced by alanines. (B, C) Positions of mutations in the Bicc1 SAM dimer model (B) and on the ML surface and EH surface (C). (D) Table summarizing, for each amino acid patch, the average number of H bonds per time frame during the MD. (E and F) Pulldown of the WT, point mutants, or 8-fold excess bpk mutant HA-tagged Bicc1 from HEK293T cell extracts by glutathione-Sepharose beads coated with a recombinant GST control or GST-Bicc1 SAM. Five percent of total cell extracts were loaded as input.

Techniques Used: Mutagenesis, Recombinant

35) Product Images from "Sorting Nexin 6 Enhances Lamin A Synthesis and Incorporation into the Nuclear Envelope"

Article Title: Sorting Nexin 6 Enhances Lamin A Synthesis and Incorporation into the Nuclear Envelope

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115571

SNX6 interacts with lamin A/C in vitro and in vivo . ( A ) Cell extracts from U2OS cells overexpressing HA-lamin A (left) or mouse smooth muscle cells (SMCs) expressing endogenous lamin A (right) were subjected to pull-down with GST-SNX6 or GST alone. Pelleted material was probed by immunoblot with the indicated antibodies. Input lane corresponds to an aliquot of the total protein mixture before each pull down experiment. ( B ) In vivo interaction between SNX6 and lamin A was quantified by fluorescence resonance energy transfer (FRET) using the acceptor photobleaching method. Data in the graph represent the mean±SE of three independent experiments. The images show a representative example of cells cotransfected with YFP-SNX6 and CFP-LMNA before and after YFP photobleaching. ( C ) U2OS cells were transiently transfected with YFP-SNX6 together with either HA-lamin A or HA alone as indicated. Cell lysates were immunoprecipitated (IP) with anti-GFP antibodies or control immunoglobulins and immunocomplexes were further analyzed by immunoblotting with anti-HA (top blot) to visualize specific interactions or with anti-GFP (bottom blot) to validate the experimental procedure. Ctrl- indicates the use of unrelated antibodies for immunoprecipitation. ( D ) Interaction between endogenous lamin A and SNX6. Cell extracts from mouse embryonic fibroblasts (MEFs) and U2OS cells (right) were immunoprecipitated with antibodies against lamin A (LMNA) or against unrelated proteins (SP1 and UCP2). Samples were analyzed by Western blot with the indicated antibodies.
Figure Legend Snippet: SNX6 interacts with lamin A/C in vitro and in vivo . ( A ) Cell extracts from U2OS cells overexpressing HA-lamin A (left) or mouse smooth muscle cells (SMCs) expressing endogenous lamin A (right) were subjected to pull-down with GST-SNX6 or GST alone. Pelleted material was probed by immunoblot with the indicated antibodies. Input lane corresponds to an aliquot of the total protein mixture before each pull down experiment. ( B ) In vivo interaction between SNX6 and lamin A was quantified by fluorescence resonance energy transfer (FRET) using the acceptor photobleaching method. Data in the graph represent the mean±SE of three independent experiments. The images show a representative example of cells cotransfected with YFP-SNX6 and CFP-LMNA before and after YFP photobleaching. ( C ) U2OS cells were transiently transfected with YFP-SNX6 together with either HA-lamin A or HA alone as indicated. Cell lysates were immunoprecipitated (IP) with anti-GFP antibodies or control immunoglobulins and immunocomplexes were further analyzed by immunoblotting with anti-HA (top blot) to visualize specific interactions or with anti-GFP (bottom blot) to validate the experimental procedure. Ctrl- indicates the use of unrelated antibodies for immunoprecipitation. ( D ) Interaction between endogenous lamin A and SNX6. Cell extracts from mouse embryonic fibroblasts (MEFs) and U2OS cells (right) were immunoprecipitated with antibodies against lamin A (LMNA) or against unrelated proteins (SP1 and UCP2). Samples were analyzed by Western blot with the indicated antibodies.

Techniques Used: In Vitro, In Vivo, Expressing, Fluorescence, Förster Resonance Energy Transfer, Transfection, Immunoprecipitation, Western Blot

36) 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

37) Product Images from "Comparative Cell Biology and Evolution of Annexins in Diplomonads"

Article Title: Comparative Cell Biology and Evolution of Annexins in Diplomonads

Journal: mSphere

doi: 10.1128/mSphere.00032-15

Membrane- and phospholipid-binding characteristics of S. salmonicida annexins. Membrane fractionation of transfected cell lines was performed to enrich for integral membrane proteins. (A) The presence of the annexins in the resulting hydrophilic (cytoplasmic) and hydrophobic (membrane) fractions was analyzed by Western blotting with the HA epitope tag. The lower two panel rows show the corresponding loading controls with the Bio-Rad stain-free TGX system. Membrane strips containing 15 biologically active lipids were used to investigate the phospholipid-binding preferences of purified recombinant annexin 3 (B), annexin 5 (C), and alpha-14 giardin (D). Abbreviations: DAG, diacylglycerol; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; PtdIns(3,5)P 3 , phosphatidylinositol 3,5-triphosphate. Molecular sizes in kilodaltons are indicated to the left of the blots.
Figure Legend Snippet: Membrane- and phospholipid-binding characteristics of S. salmonicida annexins. Membrane fractionation of transfected cell lines was performed to enrich for integral membrane proteins. (A) The presence of the annexins in the resulting hydrophilic (cytoplasmic) and hydrophobic (membrane) fractions was analyzed by Western blotting with the HA epitope tag. The lower two panel rows show the corresponding loading controls with the Bio-Rad stain-free TGX system. Membrane strips containing 15 biologically active lipids were used to investigate the phospholipid-binding preferences of purified recombinant annexin 3 (B), annexin 5 (C), and alpha-14 giardin (D). Abbreviations: DAG, diacylglycerol; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; PtdIns(3,5)P 3 , phosphatidylinositol 3,5-triphosphate. Molecular sizes in kilodaltons are indicated to the left of the blots.

Techniques Used: Binding Assay, Fractionation, Transfection, Western Blot, Staining, Purification, Recombinant

38) Product Images from "Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome"

Article Title: Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome

Journal: Nature Communications

doi: 10.1038/ncomms12497

AMPK directly regulates SirT7 phosphorylation and subcellular distribution under starvation. ( a ) 293T cells transfected with Flag-SirT7 and HA-AMPKα plasmids were treated with or without GD (4 h), followed by immunoprecipitation with FLAG-M2 beads. The precipitated proteins were analysed by western blot using anti-Flag or anti-HA antibody. ( b ) In vitro phosphorylation of SirT7 by activated AMPKα. GST-SirT7-WT or -153A mutants were expressed in E.coli and purified with GST beads. Activated Flag-AMPKα was precipitated from GD (6 h) treated Flag-AMPKα-overexpressing 293T cells using FLAG-M2 beads and eluted with Flag peptide. GST-SirT7-WT or -153A proteins were incubated with or without Flag-AMPKα in the presence or absence of ATP as indicated. The reaction product was separated by SDS-PAGE and analysed by western blot. ( c ) Similar in vitro kinase assay was performed as detailed for ( b ), except that the kinase-dead AMPKα D159A (AMPK-DN) mutant was used. ( d ) HeLa cells with AMPKα knockdown (AMPKα-Si) or control Si-RNA (Ctrl-Si) were transfected with Flag-SirT7 following GD (12 h) treatment, then immunostained with anti-Flag (red) or anti-UBF (green) antibodies (scale bar, 10 μm). Nuclei were stained with DAPI. ( e ) HeLa cells with AMPKα knockdown or control Si-RNA were treated with GD (4 h) followed by immunoprecipitation with anti-SirT7 antibody and western blot analysis. AMPK activation was confirmed by its phosphorylation at Thr-172.
Figure Legend Snippet: AMPK directly regulates SirT7 phosphorylation and subcellular distribution under starvation. ( a ) 293T cells transfected with Flag-SirT7 and HA-AMPKα plasmids were treated with or without GD (4 h), followed by immunoprecipitation with FLAG-M2 beads. The precipitated proteins were analysed by western blot using anti-Flag or anti-HA antibody. ( b ) In vitro phosphorylation of SirT7 by activated AMPKα. GST-SirT7-WT or -153A mutants were expressed in E.coli and purified with GST beads. Activated Flag-AMPKα was precipitated from GD (6 h) treated Flag-AMPKα-overexpressing 293T cells using FLAG-M2 beads and eluted with Flag peptide. GST-SirT7-WT or -153A proteins were incubated with or without Flag-AMPKα in the presence or absence of ATP as indicated. The reaction product was separated by SDS-PAGE and analysed by western blot. ( c ) Similar in vitro kinase assay was performed as detailed for ( b ), except that the kinase-dead AMPKα D159A (AMPK-DN) mutant was used. ( d ) HeLa cells with AMPKα knockdown (AMPKα-Si) or control Si-RNA (Ctrl-Si) were transfected with Flag-SirT7 following GD (12 h) treatment, then immunostained with anti-Flag (red) or anti-UBF (green) antibodies (scale bar, 10 μm). Nuclei were stained with DAPI. ( e ) HeLa cells with AMPKα knockdown or control Si-RNA were treated with GD (4 h) followed by immunoprecipitation with anti-SirT7 antibody and western blot analysis. AMPK activation was confirmed by its phosphorylation at Thr-172.

Techniques Used: Transfection, Immunoprecipitation, Western Blot, In Vitro, Purification, Incubation, SDS Page, Kinase Assay, Mutagenesis, Staining, Activation Assay

39) Product Images from "TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A"

Article Title: TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A

Journal: Scientific Reports

doi: 10.1038/srep33698

Interaction of TRIM52 with JEV NS2A protein. ( a ) 293T cells in six-well plates were co-transfected with expression plasmids encoding HA-TRIM52 plus the indicated plasmids expressing Flag-tagged JEV viral proteins. The cells were harvested at 36 hpt, and the cell lysates were prepared for Western blot analysis using the indicated antibodies. Immunoprecipitation (IP) was performed using anti-Flag affinity gel. WCL, whole cell lysates. ( b ) Co-localization of TRIM52 with NS2A. 293T cells were co-transfected with plasmids expressing HA-TRIM52 plus NS2A-Flag. The cells were fixed at 24 hpt and were subjected to immunofluorescence to detect HA-TRIM52 (green) and NS2A-Flag (red). The nuclei were stained with DAPI (blue). Images were obtained using confocal microscope (Carl Zeiss MicroImaging, Inc.). ( c ) Purified proteins of GST and GST-TRIM52 were analyzed by SDS-PAGE. ( d ) Western blot analysis of the anti-Flag affinity NS2A from 293T-NS2A cell lysates. ( e ) GST pull-down and Western blot analysis of the interaction between TRIM52 and NS2A. ( f ) IP using anti-Flag affinity gel and Western blot analysis for the interaction of NS2A with TRIM52 and RING domain deleted TRIM52 in 293T cells co-transfected with NS2A-Flag or with empty vector plus HA-TRIM52 and HA-TRIM52dR.
Figure Legend Snippet: Interaction of TRIM52 with JEV NS2A protein. ( a ) 293T cells in six-well plates were co-transfected with expression plasmids encoding HA-TRIM52 plus the indicated plasmids expressing Flag-tagged JEV viral proteins. The cells were harvested at 36 hpt, and the cell lysates were prepared for Western blot analysis using the indicated antibodies. Immunoprecipitation (IP) was performed using anti-Flag affinity gel. WCL, whole cell lysates. ( b ) Co-localization of TRIM52 with NS2A. 293T cells were co-transfected with plasmids expressing HA-TRIM52 plus NS2A-Flag. The cells were fixed at 24 hpt and were subjected to immunofluorescence to detect HA-TRIM52 (green) and NS2A-Flag (red). The nuclei were stained with DAPI (blue). Images were obtained using confocal microscope (Carl Zeiss MicroImaging, Inc.). ( c ) Purified proteins of GST and GST-TRIM52 were analyzed by SDS-PAGE. ( d ) Western blot analysis of the anti-Flag affinity NS2A from 293T-NS2A cell lysates. ( e ) GST pull-down and Western blot analysis of the interaction between TRIM52 and NS2A. ( f ) IP using anti-Flag affinity gel and Western blot analysis for the interaction of NS2A with TRIM52 and RING domain deleted TRIM52 in 293T cells co-transfected with NS2A-Flag or with empty vector plus HA-TRIM52 and HA-TRIM52dR.

Techniques Used: Transfection, Expressing, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Microscopy, Purification, SDS Page, Plasmid Preparation

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025694

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

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

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

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

Related Articles

Affinity Chromatography:

Article Title: In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1
Article Snippet: .. PN-1 was purified from bacterial lysates by affinity chromatography using glutathione-Sepharose 4B and cleaved from GST with a site-specific protease, followed by gel filtration chromatography on Sephadex G75 (GE Healthcare) ( , inset). .. The stoichiometries for WT and K7Q PN-1 binding to thrombin were determined by measuring the loss of thrombin activity on the chromogenic substrate S-2238 and gave values from 3.3 to 4.1.

Filtration:

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4
Article Snippet: .. The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin (Sigma-Aldrich, St. Louis, MI, USA) was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg (GE Healthcare) with PBS. ..

Article Title: In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1
Article Snippet: .. PN-1 was purified from bacterial lysates by affinity chromatography using glutathione-Sepharose 4B and cleaved from GST with a site-specific protease, followed by gel filtration chromatography on Sephadex G75 (GE Healthcare) ( , inset). .. The stoichiometries for WT and K7Q PN-1 binding to thrombin were determined by measuring the loss of thrombin activity on the chromogenic substrate S-2238 and gave values from 3.3 to 4.1.

Pull Down Assay:

Article Title: Plant begomoviruses subvert ubiquitination to suppress plant defenses against insect vectors
Article Snippet: .. Pull-down Assay The recombinant GST and MBP tag proteins were purified using GST- (GE Healthcare) or MBP-Trap ( New England Biolabs ) according to the manufacturer’s instructions. .. The pulled-down proteins were separated on 12% SDS-PAGE gels and detected by western blot using anti-MBP antibody (Abcam).

Chromatography:

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4
Article Snippet: .. The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin (Sigma-Aldrich, St. Louis, MI, USA) was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg (GE Healthcare) with PBS. ..

Article Title: In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1
Article Snippet: .. PN-1 was purified from bacterial lysates by affinity chromatography using glutathione-Sepharose 4B and cleaved from GST with a site-specific protease, followed by gel filtration chromatography on Sephadex G75 (GE Healthcare) ( , inset). .. The stoichiometries for WT and K7Q PN-1 binding to thrombin were determined by measuring the loss of thrombin activity on the chromogenic substrate S-2238 and gave values from 3.3 to 4.1.

Mutagenesis:

Article Title: Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion
Article Snippet: .. For GST-fused various fragments of human α-Parvin, each gene of the N-terminal domain (residues 1–89), or its mutant 5A (1–91) or the full-length (residues 1–372) of α-Parvin was subcloned into pGEX4T1 vector (GE Healthcare). .. These GST-fused α-Parvin proteins were expressed in E. coli and affinity-purified by glutathione-Sepharose 4B affinity column (GE Healthcare).

Purification:

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4
Article Snippet: .. The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin (Sigma-Aldrich, St. Louis, MI, USA) was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg (GE Healthcare) with PBS. ..

Article Title: STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer
Article Snippet: .. His-STK25, GST-GOLPH3, and GST were expressed and purified in accordance with the manufacturer’s instructions (Amersham). .. Then, 10 μg of His-STK25 was mixed with 10 μg of GST-GOLPH3 or GST and incubated with glutathione sepharose 4B beads (GE Healthcare) and Ni-NTA agarose (QIAGEN), respectively, for the GST pull-down and His-tag pull-down assays.

Article Title: Plant begomoviruses subvert ubiquitination to suppress plant defenses against insect vectors
Article Snippet: .. Pull-down Assay The recombinant GST and MBP tag proteins were purified using GST- (GE Healthcare) or MBP-Trap ( New England Biolabs ) according to the manufacturer’s instructions. .. The pulled-down proteins were separated on 12% SDS-PAGE gels and detected by western blot using anti-MBP antibody (Abcam).

Article Title: In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1
Article Snippet: .. PN-1 was purified from bacterial lysates by affinity chromatography using glutathione-Sepharose 4B and cleaved from GST with a site-specific protease, followed by gel filtration chromatography on Sephadex G75 (GE Healthcare) ( , inset). .. The stoichiometries for WT and K7Q PN-1 binding to thrombin were determined by measuring the loss of thrombin activity on the chromogenic substrate S-2238 and gave values from 3.3 to 4.1.

Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation
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). .. The purified GST-fusion proteins were desalted on Vivaspin 6 column from GE Healthcare, followed by dialysis into binding buffer (PBS, 1 mM MgCI2 , pH 7.4) to remove the free glutathione.

Incubation:

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4
Article Snippet: .. The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin (Sigma-Aldrich, St. Louis, MI, USA) was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg (GE Healthcare) with PBS. ..

Affinity Purification:

Article Title: PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)
Article Snippet: .. The truncates (Tr) Tr1, residues E22 -S263 ; Tr2, A264 -D501 ; Tr3, I508 -P723 ; Tr4, A805 -P1093 ; and Tr5, L1193 -P1455 were affinity purified using a glutathione-Sepharose 4B column (GE Healthcare, Camarillo, CA). .. The captured proteins were eluted by either on-column cleavage with AcTEV protease (Thermo Fisher Scientific, Waltham, MA) (Tr1, 2 and 4), targeting a tobacco etch virus (TEV) protease recognition site located between the GST tag and the recombinant protein, or with 20 mM glutathione (Tr3 and 5).

Recombinant:

Article Title: Plant begomoviruses subvert ubiquitination to suppress plant defenses against insect vectors
Article Snippet: .. Pull-down Assay The recombinant GST and MBP tag proteins were purified using GST- (GE Healthcare) or MBP-Trap ( New England Biolabs ) according to the manufacturer’s instructions. .. The pulled-down proteins were separated on 12% SDS-PAGE gels and detected by western blot using anti-MBP antibody (Abcam).

Plasmid Preparation:

Article Title: Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion
Article Snippet: .. For GST-fused various fragments of human α-Parvin, each gene of the N-terminal domain (residues 1–89), or its mutant 5A (1–91) or the full-length (residues 1–372) of α-Parvin was subcloned into pGEX4T1 vector (GE Healthcare). .. These GST-fused α-Parvin proteins were expressed in E. coli and affinity-purified by glutathione-Sepharose 4B affinity column (GE Healthcare).

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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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    94
    GE Healthcare glutathione sepharose 4b beads
    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