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Xic1 interacts with DDB1 and XCdt2. (A) Schematic representation of XCdt2. XCdt2 contains two WDXR motifs (gray boxes), six WD40 domains (white boxes), and a conserved arginine residue (R247) essential for DDB1 binding. (B) GST pulldown assay. GST or GST-Xic1 immobilized on <t>glutathione-Sepharose</t> beads was incubated with Xenopus interphase egg extract and immunoblotted with antibody against Xenopus DDB1 and PCNA (Western blot). GST and GST-Xic1 proteins (20% of Western blot reaction) were stained with Coomassie brilliant blue. The input (4%) is shown in lane 1. α, anti; *, nonspecific bacterial protein. (C) Coimmunoprecipitation assay. Immunoprecipitated DDB1 (IP) from the egg extract was bound to protein A beads and incubated with 35 S-labeled wild-type XCdt2 (WT), XCdt2 R247A (R247A), XCdt2 1-400 (1-400), or XCdt2 401-710 (401-710). As a control, nonspecific normal rabbit serum (NRS) was used in the place of DDB1 antiserum. Efficient immunoprecipitation of XDDB1 was confirmed by immunoblotting with anti-DDB1 antibody (top). Binding of XCdt2 proteins ( 35 S-Cdt2) was analyzed by SDS-PAGE and phosphorimaging, and 5% of the input proteins is shown (5% input). (D) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with 35 S-labeled Xic1 and subjected to SDS-PAGE and phosphorimager analysis. Efficient immunoprecipitation of XCdt2 was confirmed by immunoblotting with anti-Cdt2 antibody (top). Immunoprecipitation with normal rabbit serum (NRS) was included as a control, and input samples are indicated. (E) GST pulldown assay. Bacterially expressed GST or GST-Xic1 (5 μg) was immobilized on <t>glutathione-Sepharose</t> beads and incubated with 35 S-labeled XCdt2 proteins, as indicated. A total of 5% of the input reaction is shown. The percentage of Cdt2 bound by all GST-Xic1 proteins (% binding) is an average value obtained from 2 independent experiments.
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1) Product Images from "The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01135-09

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

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

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

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

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

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

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

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

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

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

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

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01135-09

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

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

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

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

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

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

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

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

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

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

3) Product Images from "Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface"

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface

Journal: Journal of Virology

doi: 10.1128/JVI.02606-13

hCMV IE1 exhibits two separable histone binding domains with differential specificities for H2A-H2B and H3-H4. (A) Schematic of wild-type and mutant hCMV IE1 and KSHV LANA proteins with relative locations of their CTDs. (B) Results of in vitro GST pulldown assays with acid-extracted unfractionated human histones. Empty glutathione-Sepharose beads or beads carrying GST or the indicated GST fusion proteins were reacted with acid-extracted human histones. Input and output protein samples were separated along with purified recombinant human core histones (H2A, H2B, H3, and H4 from New England BioLabs) in a 15% polyacrylamide-SDS gel and stained with Coomassie brilliant blue. The asterisk marks H1 histones. (C) Results of in vitro GST pulldown assays with acid-extracted human histones separated into H2A-H2B and H3-H4 fractions. Equal volumes of empty glutathione-Sepharose beads or beads carrying GST or the indicated GST fusion proteins were reacted with purified human H2A-H2B or H3-H4. Input (8% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue.
Figure Legend Snippet: hCMV IE1 exhibits two separable histone binding domains with differential specificities for H2A-H2B and H3-H4. (A) Schematic of wild-type and mutant hCMV IE1 and KSHV LANA proteins with relative locations of their CTDs. (B) Results of in vitro GST pulldown assays with acid-extracted unfractionated human histones. Empty glutathione-Sepharose beads or beads carrying GST or the indicated GST fusion proteins were reacted with acid-extracted human histones. Input and output protein samples were separated along with purified recombinant human core histones (H2A, H2B, H3, and H4 from New England BioLabs) in a 15% polyacrylamide-SDS gel and stained with Coomassie brilliant blue. The asterisk marks H1 histones. (C) Results of in vitro GST pulldown assays with acid-extracted human histones separated into H2A-H2B and H3-H4 fractions. Equal volumes of empty glutathione-Sepharose beads or beads carrying GST or the indicated GST fusion proteins were reacted with purified human H2A-H2B or H3-H4. Input (8% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue.

Techniques Used: Binding Assay, Mutagenesis, In Vitro, Purification, Recombinant, SDS-Gel, Staining

Alanine scanning mutagenesis identifies IE1 CTD residues critical for histone binding. (A) Presentation of wild-type (wt) and mutant IE1 476–491 protein sequences. Amino acids substituted by alanine scanning mutagenesis are highlighted. (B) Results of in vitro GST pulldown assays. Glutathione-Sepharose beads carrying GST or the indicated wild-type (wt) and mutant GST-IE1 476–491 fusion proteins (see also panel A) were reacted with acid-extracted histones. Input (14% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. (C) Quantitative assessment of GST pulldown assay results. The output bands were quantified by densitometry, and bars represent the ratios of histones H3, H2B, H2A, and H4 to GST-IE1 476–491 . Results for the CTD mutants are presented relative to those for the CTD wild-type sample present on the same gel (set to 100%).
Figure Legend Snippet: Alanine scanning mutagenesis identifies IE1 CTD residues critical for histone binding. (A) Presentation of wild-type (wt) and mutant IE1 476–491 protein sequences. Amino acids substituted by alanine scanning mutagenesis are highlighted. (B) Results of in vitro GST pulldown assays. Glutathione-Sepharose beads carrying GST or the indicated wild-type (wt) and mutant GST-IE1 476–491 fusion proteins (see also panel A) were reacted with acid-extracted histones. Input (14% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. (C) Quantitative assessment of GST pulldown assay results. The output bands were quantified by densitometry, and bars represent the ratios of histones H3, H2B, H2A, and H4 to GST-IE1 476–491 . Results for the CTD mutants are presented relative to those for the CTD wild-type sample present on the same gel (set to 100%).

Techniques Used: Mutagenesis, Binding Assay, In Vitro, Staining, GST Pulldown Assay

The IE1 and LANA CTDs compete for binding to human core histones. (A) Full-length IE1 competes with GST-LANA 5–22 for histone binding. (B) IE1 lacking the CTD (IE1 1–475 ) is less active in competing with GST-LANA 5–22 for histone binding than the full-length protein. (C) A peptide encompassing LANA 5–22 (LANA-CTD), but not a mutant peptide (LANA-CTD*), interferes with histone binding to GST-IE1 476–491 . Acid-extracted human histones were combined with solvent or with the indicated soluble IE1 proteins (A, B) or LANA peptides (C) and then subjected to in vitro GST pulldown assays with glutathione-Sepharose beads carrying GST-LANA 5–22 (A, B) or GST-IE1 476–491 (C). Input and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. See also Fig. S3 in the supplemental material.
Figure Legend Snippet: The IE1 and LANA CTDs compete for binding to human core histones. (A) Full-length IE1 competes with GST-LANA 5–22 for histone binding. (B) IE1 lacking the CTD (IE1 1–475 ) is less active in competing with GST-LANA 5–22 for histone binding than the full-length protein. (C) A peptide encompassing LANA 5–22 (LANA-CTD), but not a mutant peptide (LANA-CTD*), interferes with histone binding to GST-IE1 476–491 . Acid-extracted human histones were combined with solvent or with the indicated soluble IE1 proteins (A, B) or LANA peptides (C) and then subjected to in vitro GST pulldown assays with glutathione-Sepharose beads carrying GST-LANA 5–22 (A, B) or GST-IE1 476–491 (C). Input and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. See also Fig. S3 in the supplemental material.

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

hCMV IE1 interacts with human nucleosomes and all four core histones in a nucleic acid-independent fashion. (A) Results of coimmunoprecipitations from plasmid-transfected cells. H1299 cells were transfected with plasmids encoding the indicated HA-tagged viral proteins or with an empty vector (w/o). Cell extracts were combined with nucleosomes derived from MNase-digested human cell nuclei. Samples were subjected to immunoprecipitation using anti-HA or anti-Flag agarose. Input and output protein samples were separated in 10 or 15% polyacrylamide-SDS gels, and HA-tagged proteins and individual core histones were detected by immunoblotting. See also Fig. S1 in the supplemental material. (B) Results of coimmunoprecipitations from hCMV-infected cells. Following infection of MRC-5 cells with TNwt or TNdlIE1 viruses (3 PFU/cell for 72 h), cells were fixed with formaldehyde and cell extracts were sonicated to solubilize nucleosomes. Samples were subjected to immunoprecipitation using rabbit antibodies to histone H3 or nonspecific rabbit IgG (rbIgG). Input and output protein samples were separated in 15% polyacrylamide-SDS gels, and the IE1 protein and histone H3 were detected by immunoblotting. (C) Results of in vitro GST pulldown assays. Equal volumes of empty glutathione-Sepharose beads or beads carrying GST or GST-IE1 were reacted with acid-extracted human histones in the absence or presence of DNase I and RNase A. Input (8% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue.
Figure Legend Snippet: hCMV IE1 interacts with human nucleosomes and all four core histones in a nucleic acid-independent fashion. (A) Results of coimmunoprecipitations from plasmid-transfected cells. H1299 cells were transfected with plasmids encoding the indicated HA-tagged viral proteins or with an empty vector (w/o). Cell extracts were combined with nucleosomes derived from MNase-digested human cell nuclei. Samples were subjected to immunoprecipitation using anti-HA or anti-Flag agarose. Input and output protein samples were separated in 10 or 15% polyacrylamide-SDS gels, and HA-tagged proteins and individual core histones were detected by immunoblotting. See also Fig. S1 in the supplemental material. (B) Results of coimmunoprecipitations from hCMV-infected cells. Following infection of MRC-5 cells with TNwt or TNdlIE1 viruses (3 PFU/cell for 72 h), cells were fixed with formaldehyde and cell extracts were sonicated to solubilize nucleosomes. Samples were subjected to immunoprecipitation using rabbit antibodies to histone H3 or nonspecific rabbit IgG (rbIgG). Input and output protein samples were separated in 15% polyacrylamide-SDS gels, and the IE1 protein and histone H3 were detected by immunoblotting. (C) Results of in vitro GST pulldown assays. Equal volumes of empty glutathione-Sepharose beads or beads carrying GST or GST-IE1 were reacted with acid-extracted human histones in the absence or presence of DNase I and RNase A. Input (8% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue.

Techniques Used: Plasmid Preparation, Transfection, Derivative Assay, Immunoprecipitation, Infection, Sonication, In Vitro, Staining

Site-directed mutagenesis identifies human H2A residues critical for interaction with IE1. (A) H2A residues targeted by mutagenesis and evaluated for contributions to IE1 binding. The complete sequence of human histone H2A.2 (H2A type 1B/E) is shown, with acidic residues in bold letters and amino acids that form the acidic pocket underlined. (B) Results of in vitro GST pulldown assays performed with the IE1 CTD. Empty glutathione-Sepharose beads or beads carrying GST-IE1 476–491 were reacted with acid-extracted human histones from H1299 cells transfected with an empty vector (w/o) or plasmids encoding the indicated Flag-tagged H2A proteins. Input and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. Flag-H2A proteins were detected by immunoblotting. (C) Results of coimmunoprecipitations performed with full-length IE1 protein. H1299 cells were simultaneously transfected with an empty vector (w/o) or plasmids encoding the indicated wild-type (wt) and mutant Flag-H2A proteins and plasmids encoding either HA-tagged full-length IE1 or HA-IE1 1–475 . Cells were fixed with formaldehyde, and cell extracts were sonicated to solubilize nucleosomes. Samples were subjected to immunoprecipitation using anti-HA or mouse IgG (mIgG) agarose. Input and output protein samples were separated in 10 or 15% polyacrylamide-SDS gels, and Flag- and HA-tagged proteins were detected by immunoblotting. (D) Quantitative assessment of results. The α-Flag bands shown in panel C were quantified by densitometry, and bars represent the ratios of output to input signal intensities r elative to that of the H2A wild-type sample present on the same blot (whose intensity was set to 100%).
Figure Legend Snippet: Site-directed mutagenesis identifies human H2A residues critical for interaction with IE1. (A) H2A residues targeted by mutagenesis and evaluated for contributions to IE1 binding. The complete sequence of human histone H2A.2 (H2A type 1B/E) is shown, with acidic residues in bold letters and amino acids that form the acidic pocket underlined. (B) Results of in vitro GST pulldown assays performed with the IE1 CTD. Empty glutathione-Sepharose beads or beads carrying GST-IE1 476–491 were reacted with acid-extracted human histones from H1299 cells transfected with an empty vector (w/o) or plasmids encoding the indicated Flag-tagged H2A proteins. Input and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. Flag-H2A proteins were detected by immunoblotting. (C) Results of coimmunoprecipitations performed with full-length IE1 protein. H1299 cells were simultaneously transfected with an empty vector (w/o) or plasmids encoding the indicated wild-type (wt) and mutant Flag-H2A proteins and plasmids encoding either HA-tagged full-length IE1 or HA-IE1 1–475 . Cells were fixed with formaldehyde, and cell extracts were sonicated to solubilize nucleosomes. Samples were subjected to immunoprecipitation using anti-HA or mouse IgG (mIgG) agarose. Input and output protein samples were separated in 10 or 15% polyacrylamide-SDS gels, and Flag- and HA-tagged proteins were detected by immunoblotting. (D) Quantitative assessment of results. The α-Flag bands shown in panel C were quantified by densitometry, and bars represent the ratios of output to input signal intensities r elative to that of the H2A wild-type sample present on the same blot (whose intensity was set to 100%).

Techniques Used: Mutagenesis, Binding Assay, Sequencing, In Vitro, Transfection, Plasmid Preparation, Staining, Sonication, Immunoprecipitation

4) Product Images from "Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface"

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface

Journal: Journal of Virology

doi: 10.1128/JVI.02606-13

hCMV IE1 exhibits two separable histone binding domains with differential specificities for H2A-H2B and H3-H4. (A) Schematic of wild-type and mutant hCMV IE1 and KSHV LANA proteins with relative locations of their CTDs. (B) Results of in vitro GST pulldown assays with acid-extracted unfractionated human histones. Empty glutathione-Sepharose beads or beads carrying GST or the indicated GST fusion proteins were reacted with acid-extracted human histones. Input and output protein samples were separated along with purified recombinant human core histones (H2A, H2B, H3, and H4 from New England BioLabs) in a 15% polyacrylamide-SDS gel and stained with Coomassie brilliant blue. The asterisk marks H1 histones. (C) Results of in vitro GST pulldown assays with acid-extracted human histones separated into H2A-H2B and H3-H4 fractions. Equal volumes of empty glutathione-Sepharose beads or beads carrying GST or the indicated GST fusion proteins were reacted with purified human H2A-H2B or H3-H4. Input (8% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue.
Figure Legend Snippet: hCMV IE1 exhibits two separable histone binding domains with differential specificities for H2A-H2B and H3-H4. (A) Schematic of wild-type and mutant hCMV IE1 and KSHV LANA proteins with relative locations of their CTDs. (B) Results of in vitro GST pulldown assays with acid-extracted unfractionated human histones. Empty glutathione-Sepharose beads or beads carrying GST or the indicated GST fusion proteins were reacted with acid-extracted human histones. Input and output protein samples were separated along with purified recombinant human core histones (H2A, H2B, H3, and H4 from New England BioLabs) in a 15% polyacrylamide-SDS gel and stained with Coomassie brilliant blue. The asterisk marks H1 histones. (C) Results of in vitro GST pulldown assays with acid-extracted human histones separated into H2A-H2B and H3-H4 fractions. Equal volumes of empty glutathione-Sepharose beads or beads carrying GST or the indicated GST fusion proteins were reacted with purified human H2A-H2B or H3-H4. Input (8% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue.

Techniques Used: Binding Assay, Mutagenesis, In Vitro, Purification, Recombinant, SDS-Gel, Staining

Alanine scanning mutagenesis identifies IE1 CTD residues critical for histone binding. (A) Presentation of wild-type (wt) and mutant IE1 476–491 protein sequences. Amino acids substituted by alanine scanning mutagenesis are highlighted. (B) Results of in vitro GST pulldown assays. Glutathione-Sepharose beads carrying GST or the indicated wild-type (wt) and mutant GST-IE1 476–491 fusion proteins (see also panel A) were reacted with acid-extracted histones. Input (14% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. (C) Quantitative assessment of GST pulldown assay results. The output bands were quantified by densitometry, and bars represent the ratios of histones H3, H2B, H2A, and H4 to GST-IE1 476–491 . Results for the CTD mutants are presented relative to those for the CTD wild-type sample present on the same gel (set to 100%).
Figure Legend Snippet: Alanine scanning mutagenesis identifies IE1 CTD residues critical for histone binding. (A) Presentation of wild-type (wt) and mutant IE1 476–491 protein sequences. Amino acids substituted by alanine scanning mutagenesis are highlighted. (B) Results of in vitro GST pulldown assays. Glutathione-Sepharose beads carrying GST or the indicated wild-type (wt) and mutant GST-IE1 476–491 fusion proteins (see also panel A) were reacted with acid-extracted histones. Input (14% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. (C) Quantitative assessment of GST pulldown assay results. The output bands were quantified by densitometry, and bars represent the ratios of histones H3, H2B, H2A, and H4 to GST-IE1 476–491 . Results for the CTD mutants are presented relative to those for the CTD wild-type sample present on the same gel (set to 100%).

Techniques Used: Mutagenesis, Binding Assay, In Vitro, Staining, GST Pulldown Assay

The IE1 and LANA CTDs compete for binding to human core histones. (A) Full-length IE1 competes with GST-LANA 5–22 for histone binding. (B) IE1 lacking the CTD (IE1 1–475 ) is less active in competing with GST-LANA 5–22 for histone binding than the full-length protein. (C) A peptide encompassing LANA 5–22 (LANA-CTD), but not a mutant peptide (LANA-CTD*), interferes with histone binding to GST-IE1 476–491 . Acid-extracted human histones were combined with solvent or with the indicated soluble IE1 proteins (A, B) or LANA peptides (C) and then subjected to in vitro GST pulldown assays with glutathione-Sepharose beads carrying GST-LANA 5–22 (A, B) or GST-IE1 476–491 (C). Input and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. See also Fig. S3 in the supplemental material.
Figure Legend Snippet: The IE1 and LANA CTDs compete for binding to human core histones. (A) Full-length IE1 competes with GST-LANA 5–22 for histone binding. (B) IE1 lacking the CTD (IE1 1–475 ) is less active in competing with GST-LANA 5–22 for histone binding than the full-length protein. (C) A peptide encompassing LANA 5–22 (LANA-CTD), but not a mutant peptide (LANA-CTD*), interferes with histone binding to GST-IE1 476–491 . Acid-extracted human histones were combined with solvent or with the indicated soluble IE1 proteins (A, B) or LANA peptides (C) and then subjected to in vitro GST pulldown assays with glutathione-Sepharose beads carrying GST-LANA 5–22 (A, B) or GST-IE1 476–491 (C). Input and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. See also Fig. S3 in the supplemental material.

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

hCMV IE1 interacts with human nucleosomes and all four core histones in a nucleic acid-independent fashion. (A) Results of coimmunoprecipitations from plasmid-transfected cells. H1299 cells were transfected with plasmids encoding the indicated HA-tagged viral proteins or with an empty vector (w/o). Cell extracts were combined with nucleosomes derived from MNase-digested human cell nuclei. Samples were subjected to immunoprecipitation using anti-HA or anti-Flag agarose. Input and output protein samples were separated in 10 or 15% polyacrylamide-SDS gels, and HA-tagged proteins and individual core histones were detected by immunoblotting. See also Fig. S1 in the supplemental material. (B) Results of coimmunoprecipitations from hCMV-infected cells. Following infection of MRC-5 cells with TNwt or TNdlIE1 viruses (3 PFU/cell for 72 h), cells were fixed with formaldehyde and cell extracts were sonicated to solubilize nucleosomes. Samples were subjected to immunoprecipitation using rabbit antibodies to histone H3 or nonspecific rabbit IgG (rbIgG). Input and output protein samples were separated in 15% polyacrylamide-SDS gels, and the IE1 protein and histone H3 were detected by immunoblotting. (C) Results of in vitro GST pulldown assays. Equal volumes of empty glutathione-Sepharose beads or beads carrying GST or GST-IE1 were reacted with acid-extracted human histones in the absence or presence of DNase I and RNase A. Input (8% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue.
Figure Legend Snippet: hCMV IE1 interacts with human nucleosomes and all four core histones in a nucleic acid-independent fashion. (A) Results of coimmunoprecipitations from plasmid-transfected cells. H1299 cells were transfected with plasmids encoding the indicated HA-tagged viral proteins or with an empty vector (w/o). Cell extracts were combined with nucleosomes derived from MNase-digested human cell nuclei. Samples were subjected to immunoprecipitation using anti-HA or anti-Flag agarose. Input and output protein samples were separated in 10 or 15% polyacrylamide-SDS gels, and HA-tagged proteins and individual core histones were detected by immunoblotting. See also Fig. S1 in the supplemental material. (B) Results of coimmunoprecipitations from hCMV-infected cells. Following infection of MRC-5 cells with TNwt or TNdlIE1 viruses (3 PFU/cell for 72 h), cells were fixed with formaldehyde and cell extracts were sonicated to solubilize nucleosomes. Samples were subjected to immunoprecipitation using rabbit antibodies to histone H3 or nonspecific rabbit IgG (rbIgG). Input and output protein samples were separated in 15% polyacrylamide-SDS gels, and the IE1 protein and histone H3 were detected by immunoblotting. (C) Results of in vitro GST pulldown assays. Equal volumes of empty glutathione-Sepharose beads or beads carrying GST or GST-IE1 were reacted with acid-extracted human histones in the absence or presence of DNase I and RNase A. Input (8% of output) and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue.

Techniques Used: Plasmid Preparation, Transfection, Derivative Assay, Immunoprecipitation, Infection, Sonication, In Vitro, Staining

Site-directed mutagenesis identifies human H2A residues critical for interaction with IE1. (A) H2A residues targeted by mutagenesis and evaluated for contributions to IE1 binding. The complete sequence of human histone H2A.2 (H2A type 1B/E) is shown, with acidic residues in bold letters and amino acids that form the acidic pocket underlined. (B) Results of in vitro GST pulldown assays performed with the IE1 CTD. Empty glutathione-Sepharose beads or beads carrying GST-IE1 476–491 were reacted with acid-extracted human histones from H1299 cells transfected with an empty vector (w/o) or plasmids encoding the indicated Flag-tagged H2A proteins. Input and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. Flag-H2A proteins were detected by immunoblotting. (C) Results of coimmunoprecipitations performed with full-length IE1 protein. H1299 cells were simultaneously transfected with an empty vector (w/o) or plasmids encoding the indicated wild-type (wt) and mutant Flag-H2A proteins and plasmids encoding either HA-tagged full-length IE1 or HA-IE1 1–475 . Cells were fixed with formaldehyde, and cell extracts were sonicated to solubilize nucleosomes. Samples were subjected to immunoprecipitation using anti-HA or mouse IgG (mIgG) agarose. Input and output protein samples were separated in 10 or 15% polyacrylamide-SDS gels, and Flag- and HA-tagged proteins were detected by immunoblotting. (D) Quantitative assessment of results. The α-Flag bands shown in panel C were quantified by densitometry, and bars represent the ratios of output to input signal intensities r elative to that of the H2A wild-type sample present on the same blot (whose intensity was set to 100%).
Figure Legend Snippet: Site-directed mutagenesis identifies human H2A residues critical for interaction with IE1. (A) H2A residues targeted by mutagenesis and evaluated for contributions to IE1 binding. The complete sequence of human histone H2A.2 (H2A type 1B/E) is shown, with acidic residues in bold letters and amino acids that form the acidic pocket underlined. (B) Results of in vitro GST pulldown assays performed with the IE1 CTD. Empty glutathione-Sepharose beads or beads carrying GST-IE1 476–491 were reacted with acid-extracted human histones from H1299 cells transfected with an empty vector (w/o) or plasmids encoding the indicated Flag-tagged H2A proteins. Input and output protein samples were separated in 15% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. Flag-H2A proteins were detected by immunoblotting. (C) Results of coimmunoprecipitations performed with full-length IE1 protein. H1299 cells were simultaneously transfected with an empty vector (w/o) or plasmids encoding the indicated wild-type (wt) and mutant Flag-H2A proteins and plasmids encoding either HA-tagged full-length IE1 or HA-IE1 1–475 . Cells were fixed with formaldehyde, and cell extracts were sonicated to solubilize nucleosomes. Samples were subjected to immunoprecipitation using anti-HA or mouse IgG (mIgG) agarose. Input and output protein samples were separated in 10 or 15% polyacrylamide-SDS gels, and Flag- and HA-tagged proteins were detected by immunoblotting. (D) Quantitative assessment of results. The α-Flag bands shown in panel C were quantified by densitometry, and bars represent the ratios of output to input signal intensities r elative to that of the H2A wild-type sample present on the same blot (whose intensity was set to 100%).

Techniques Used: Mutagenesis, Binding Assay, Sequencing, In Vitro, Transfection, Plasmid Preparation, Staining, Sonication, Immunoprecipitation

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

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01135-09

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

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

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

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

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

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

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

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

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

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

6) Product Images from "Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13"

Article Title: Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.11.4824-4834.2004

Myc-Kap13 interacts in vivo with Pax6 but not with the Pax6 mutant (Mut) lacking regions 208 to 214 and 261 to 267. Lysates from cells cotransfected with constructs for expression of Myc-Kap13 and GFP-Pax6 were used for coimmunoprecipitation. (A) Wild-type (WT) and mutant Pax6 were immunoprecipitated equally well by the anti-Pax6 monoclonal antibody immobilized on protein G Sepharose beads. (B) Kap13 coimmunoprecipitated with wild-type Pax6 but not with the mutant Pax6. (C) Kap13 was expressed equally in both lysates.
Figure Legend Snippet: Myc-Kap13 interacts in vivo with Pax6 but not with the Pax6 mutant (Mut) lacking regions 208 to 214 and 261 to 267. Lysates from cells cotransfected with constructs for expression of Myc-Kap13 and GFP-Pax6 were used for coimmunoprecipitation. (A) Wild-type (WT) and mutant Pax6 were immunoprecipitated equally well by the anti-Pax6 monoclonal antibody immobilized on protein G Sepharose beads. (B) Kap13 coimmunoprecipitated with wild-type Pax6 but not with the mutant Pax6. (C) Kap13 was expressed equally in both lysates.

Techniques Used: In Vivo, Mutagenesis, Construct, Expressing, Immunoprecipitation

Binding of Pax6 to Kap13 is reduced when either basic cluster of Pax6, located at either end of the homeodomain, is deleted. Pax6 constructs lacking nucleotides that encode residues 208 to 214 (denoted ΔN) or 261 to 267 (ΔC) were expressed in E. coli . These constructs along with wild-type Pax6 (WT) were expressed as GST fusions, purified, immobilized on glutathione-Sepharose beads, and incubated with recombinant Kap13. (A) Pax6 has reduced binding to Kap13 when these regions are deleted (compare lanes 2, 3, and 4 to lane 1). Lane 5, Kap13 does not bind to the control GST-GFP construct. (B) The deletion of amino acids 208 to 214 decreases binding to approximately 60% compared to that of the wild type. Deletion of amino acids 261 to 267 leads to an even stronger reduction, as does the deletion of both regions. Error bars indicate standard errors of the means for three independent experiments.
Figure Legend Snippet: Binding of Pax6 to Kap13 is reduced when either basic cluster of Pax6, located at either end of the homeodomain, is deleted. Pax6 constructs lacking nucleotides that encode residues 208 to 214 (denoted ΔN) or 261 to 267 (ΔC) were expressed in E. coli . These constructs along with wild-type Pax6 (WT) were expressed as GST fusions, purified, immobilized on glutathione-Sepharose beads, and incubated with recombinant Kap13. (A) Pax6 has reduced binding to Kap13 when these regions are deleted (compare lanes 2, 3, and 4 to lane 1). Lane 5, Kap13 does not bind to the control GST-GFP construct. (B) The deletion of amino acids 208 to 214 decreases binding to approximately 60% compared to that of the wild type. Deletion of amino acids 261 to 267 leads to an even stronger reduction, as does the deletion of both regions. Error bars indicate standard errors of the means for three independent experiments.

Techniques Used: Binding Assay, Construct, Purification, Incubation, Recombinant

Kap13 binds Pax3, Pax6, and Crx (lanes 1 to 3) but not Six3, Prh, or GST-GFP (lanes 4 to 6) in a GST pull-down assay. The homeodomain factors were expressed as GST fusions in E. coli , purified, and immobilized on glutathione-Sepharose. Purified recombinant Kap13 was incubated with the immobilized homeodomain factors. The proteins retained on the beads after washing were analyzed by SDS-PAGE followed by Coomassie staining. Lane 7 shows 50% of the Kap13 that was used in lanes 1 to 6.
Figure Legend Snippet: Kap13 binds Pax3, Pax6, and Crx (lanes 1 to 3) but not Six3, Prh, or GST-GFP (lanes 4 to 6) in a GST pull-down assay. The homeodomain factors were expressed as GST fusions in E. coli , purified, and immobilized on glutathione-Sepharose. Purified recombinant Kap13 was incubated with the immobilized homeodomain factors. The proteins retained on the beads after washing were analyzed by SDS-PAGE followed by Coomassie staining. Lane 7 shows 50% of the Kap13 that was used in lanes 1 to 6.

Techniques Used: Pull Down Assay, Purification, Recombinant, Incubation, SDS Page, Staining

Immobilized Pax6 retains Kap13 from a bacterial lysate, and the binding is abolished by RanGTP. GST-Pax6 purified from E. coli was immobilized on glutathione-Sepharose beads and incubated with lysate from E. coli that expressed Kap13. The bound proteins were eluted with SDS-PAGE sample buffer and were subjected to SDS-PAGE followed by Coomassie staining. The lysate is shown in lane 2; Kap13 cannot be distinguished from the bacterial proteins. The incubation was done in the absence (lane 4) or presence (lane 5) of RanGTP. Lane 3 demonstrates that the control construct GST-GFP does not bind Kap13; lane 1 illustrates Kap13 purified from a bacterial lysate similar to that shown in lane 2.
Figure Legend Snippet: Immobilized Pax6 retains Kap13 from a bacterial lysate, and the binding is abolished by RanGTP. GST-Pax6 purified from E. coli was immobilized on glutathione-Sepharose beads and incubated with lysate from E. coli that expressed Kap13. The bound proteins were eluted with SDS-PAGE sample buffer and were subjected to SDS-PAGE followed by Coomassie staining. The lysate is shown in lane 2; Kap13 cannot be distinguished from the bacterial proteins. The incubation was done in the absence (lane 4) or presence (lane 5) of RanGTP. Lane 3 demonstrates that the control construct GST-GFP does not bind Kap13; lane 1 illustrates Kap13 purified from a bacterial lysate similar to that shown in lane 2.

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

The Pax6 residues 208 to 288 are necessary and sufficient for binding to Kap13. (A) Yeast two-hybrid interaction screens using Kap13 and deletion constructs of Pax6. The interaction was measured by using a β-galactosidase assay. The homeodomain (210 to 269) is represented by a thicker line in the left diagram. The minimal segment that interacts with Kap13 spans amino acids 208 to 288. VP16, which is fused to all constructs, does not produce any transactivation by itself. (B) GST-GFP-Pax6 segments were expressed in E. coli , immobilized on glutathione-Sepharose, and incubated with Kap13 expressed and purified from E. coli . The proteins retained on the beads were analyzed by SDS-PAGE followed by Coomassie staining. The minimal Pax6 segment that binds Kap13 is 208 to 288 (lanes 3). Lane 6 shows 50% of the amount of Kap13 incubated with the Pax6 segments.
Figure Legend Snippet: The Pax6 residues 208 to 288 are necessary and sufficient for binding to Kap13. (A) Yeast two-hybrid interaction screens using Kap13 and deletion constructs of Pax6. The interaction was measured by using a β-galactosidase assay. The homeodomain (210 to 269) is represented by a thicker line in the left diagram. The minimal segment that interacts with Kap13 spans amino acids 208 to 288. VP16, which is fused to all constructs, does not produce any transactivation by itself. (B) GST-GFP-Pax6 segments were expressed in E. coli , immobilized on glutathione-Sepharose, and incubated with Kap13 expressed and purified from E. coli . The proteins retained on the beads were analyzed by SDS-PAGE followed by Coomassie staining. The minimal Pax6 segment that binds Kap13 is 208 to 288 (lanes 3). Lane 6 shows 50% of the amount of Kap13 incubated with the Pax6 segments.

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

7) Product Images from "Inhibition of PACT-Mediated Activation of PKR by the Herpes Simplex Virus Type 1 Us11 Protein"

Article Title: Inhibition of PACT-Mediated Activation of PKR by the Herpes Simplex Virus Type 1 Us11 Protein

Journal: Journal of Virology

doi: 10.1128/JVI.76.21.11054-11064.2002

Us11C does not displace MBP-3 from PKR. Shown are the effects of increasing concentrations of purified Us11C on MBP-3 interaction with PKR. PKR was immunoprecipitated from pcDNA3-PKR-FLAG-transfected HT1080 cells by using anti-FLAG-agarose. After changing to a low-salt buffer, PKR was eluted off the beads with excess FLAG peptide. Purified Us11C and then purified PKR were added to amylose resin-immobilized MBP-3. The proteins were incubated for 1 h and washed extensively with low-salt buffer. The proteins interacting with MBP-3 were analyzed by Western blotting for PKR using FLAG antibody. Lanes 1 to 6, PKR; lanes 2 to 6, 1.0 μg of purified MBP-3; lane 3, 0.1 μg of purified Us11C; lane 4, 0.3 μg of purified Us11C; lane 5, 1.0 μg of purified Us11C; lane 6, 2.4 μg of purified Us11C. The bottom panel shows the stripped Western blot reprobed with anti-PACT domain 3 antibody.
Figure Legend Snippet: Us11C does not displace MBP-3 from PKR. Shown are the effects of increasing concentrations of purified Us11C on MBP-3 interaction with PKR. PKR was immunoprecipitated from pcDNA3-PKR-FLAG-transfected HT1080 cells by using anti-FLAG-agarose. After changing to a low-salt buffer, PKR was eluted off the beads with excess FLAG peptide. Purified Us11C and then purified PKR were added to amylose resin-immobilized MBP-3. The proteins were incubated for 1 h and washed extensively with low-salt buffer. The proteins interacting with MBP-3 were analyzed by Western blotting for PKR using FLAG antibody. Lanes 1 to 6, PKR; lanes 2 to 6, 1.0 μg of purified MBP-3; lane 3, 0.1 μg of purified Us11C; lane 4, 0.3 μg of purified Us11C; lane 5, 1.0 μg of purified Us11C; lane 6, 2.4 μg of purified Us11C. The bottom panel shows the stripped Western blot reprobed with anti-PACT domain 3 antibody.

Techniques Used: Purification, Immunoprecipitation, Transfection, Incubation, Western Blot

Us11C does not prevent PACT binding to PKR. Shown are the effects of increasing concentrations of purified Us11C on wt PACT interaction with PKR. PKR was immunoprecipitated from an extract of HT1080 cells treated with beta interferon by using PKR monoclonal antibody/protein G-Sepharose. The final two washes of immobilized PKR were done with a low-salt buffer. Us11C and then PACT were added to the immobilized PKR, followed by incubation for 15 min and then extensive washing with buffer. PACT interaction with PKR was analyzed by Western blotting using a domain 3-specific PACT antibody. All lanes contained PKR and lanes 2 to 5 contained 1 μg of PACT. In addition, 0.3 (lane 3), 1.0 (lane 4), and 2.4 (lane 5) μg of Us11C were added. The bottom panel shows the stripped Western blot reprobed with anti-PKR antibody.
Figure Legend Snippet: Us11C does not prevent PACT binding to PKR. Shown are the effects of increasing concentrations of purified Us11C on wt PACT interaction with PKR. PKR was immunoprecipitated from an extract of HT1080 cells treated with beta interferon by using PKR monoclonal antibody/protein G-Sepharose. The final two washes of immobilized PKR were done with a low-salt buffer. Us11C and then PACT were added to the immobilized PKR, followed by incubation for 15 min and then extensive washing with buffer. PACT interaction with PKR was analyzed by Western blotting using a domain 3-specific PACT antibody. All lanes contained PKR and lanes 2 to 5 contained 1 μg of PACT. In addition, 0.3 (lane 3), 1.0 (lane 4), and 2.4 (lane 5) μg of Us11C were added. The bottom panel shows the stripped Western blot reprobed with anti-PKR antibody.

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

Us11C binds to PKR residues 1 to 170. GST pulldown assays were used to measure the binding of GST-tagged Us11C to PKR and PKR deletion mutants. FLAG-tagged PKR and PKR mutants were transfected into HT1080 cells, and cell extracts were prepared. The cell extracts were mixed with either purified GST or GST-Us11C protein in binding buffer, and GST-containing protein was pulled down using glutathione-Sepharose 4B. The PKR-FLAG protein constructs interacting with the GST-containing protein were analyzed by Western blotting with FLAG antibody. In panels B to D, the amount of extracts used was twice that used in panel A. (A) Input wt PKR and mutant proteins used to measure Us11C binding. The major band in each lane shows the position of the protein. (B to D) PKR mutant proteins interacting with GST-Us11C (B), GST (C), and GST-Us11N (D). Lanes 1, full-length PKR 1-551 (K296R); lanes 2, PKR 1-170; lanes 3, PKR 171-551; lanes 4, PKR 360-551.
Figure Legend Snippet: Us11C binds to PKR residues 1 to 170. GST pulldown assays were used to measure the binding of GST-tagged Us11C to PKR and PKR deletion mutants. FLAG-tagged PKR and PKR mutants were transfected into HT1080 cells, and cell extracts were prepared. The cell extracts were mixed with either purified GST or GST-Us11C protein in binding buffer, and GST-containing protein was pulled down using glutathione-Sepharose 4B. The PKR-FLAG protein constructs interacting with the GST-containing protein were analyzed by Western blotting with FLAG antibody. In panels B to D, the amount of extracts used was twice that used in panel A. (A) Input wt PKR and mutant proteins used to measure Us11C binding. The major band in each lane shows the position of the protein. (B to D) PKR mutant proteins interacting with GST-Us11C (B), GST (C), and GST-Us11N (D). Lanes 1, full-length PKR 1-551 (K296R); lanes 2, PKR 1-170; lanes 3, PKR 171-551; lanes 4, PKR 360-551.

Techniques Used: Binding Assay, Transfection, Purification, Construct, Western Blot, Mutagenesis

In vitro translated PACTΔ3, but not Δ1, Δ2, or Δ1,2, bind Us11C. GST pulldown assays were used to measure the binding of PACT and PACT mutants to GST-tagged Us11C. 35 S-labeled wt PACT and PACT mutants were translated in vitro. Three microliters of the reticulocyte lysate containing PACT or PACT mutant was mixed with 1 μg of GST or GST-Us11C in binding buffer. Glutathione-Sepharose 4B was used to pull down GST-containing protein, and the proteins interacting with it were detected by gel electrophoresis and autoradiography. (A) Maps of human wt PACT protein and deletion constructs. Wt PACT has three domains: PKR interaction domains 1 and 2 and PKR activation domain 3. The small numbers on top indicate amino acid residue numbers. PACTΔ1 is missing PACT amino acid residues 35 to 99, Δ2 is missing PACT amino acid residues 127 to 192, Δ3 is missing PACT amino acid residues 240 to 305, and Δ1,2 is missing PACT amino acid residues 35 to 99 and 127 to 192. (B) Proteins that were translated in vitro (1 μl of lysate/lane). Lane 1, pcDNA3 vector; lane 2, wt PACT; lane 3, PACTΔ1; lane 4, PACTΔ2; lane 5, PACTΔ3; lane 6, PACTΔ1,2. (C) Proteins that interacted with the GST-tagged protein. Lane 1, GST and wt PACT; lanes 2 to 6, GST-US11C; lane 2, wt PACT; lane 3, PACTΔ1; lane 4, PACTΔ2; lane 5, PACTΔ3; lane 6, PACTΔ1,2.
Figure Legend Snippet: In vitro translated PACTΔ3, but not Δ1, Δ2, or Δ1,2, bind Us11C. GST pulldown assays were used to measure the binding of PACT and PACT mutants to GST-tagged Us11C. 35 S-labeled wt PACT and PACT mutants were translated in vitro. Three microliters of the reticulocyte lysate containing PACT or PACT mutant was mixed with 1 μg of GST or GST-Us11C in binding buffer. Glutathione-Sepharose 4B was used to pull down GST-containing protein, and the proteins interacting with it were detected by gel electrophoresis and autoradiography. (A) Maps of human wt PACT protein and deletion constructs. Wt PACT has three domains: PKR interaction domains 1 and 2 and PKR activation domain 3. The small numbers on top indicate amino acid residue numbers. PACTΔ1 is missing PACT amino acid residues 35 to 99, Δ2 is missing PACT amino acid residues 127 to 192, Δ3 is missing PACT amino acid residues 240 to 305, and Δ1,2 is missing PACT amino acid residues 35 to 99 and 127 to 192. (B) Proteins that were translated in vitro (1 μl of lysate/lane). Lane 1, pcDNA3 vector; lane 2, wt PACT; lane 3, PACTΔ1; lane 4, PACTΔ2; lane 5, PACTΔ3; lane 6, PACTΔ1,2. (C) Proteins that interacted with the GST-tagged protein. Lane 1, GST and wt PACT; lanes 2 to 6, GST-US11C; lane 2, wt PACT; lane 3, PACTΔ1; lane 4, PACTΔ2; lane 5, PACTΔ3; lane 6, PACTΔ1,2.

Techniques Used: In Vitro, Binding Assay, Labeling, Mutagenesis, Nucleic Acid Electrophoresis, Autoradiography, Construct, Activation Assay, Plasmid Preparation

Bacterially expressed PACT and PACTΔ3, but not Δ1,2, Δ1, or MBP-3, bind Us11C. GST pulldown assays were used to measure the binding of PACT and PACT mutants to GST-tagged Us11C. One microgram of purified PACT or PACT mutant protein was mixed with 1 μg of purified GST-Us11C in high-salt buffer. Glutathione-Sepharose 4B was used to pull down GST-Us11C, and the proteins interacting with it were analyzed. (A) Purified PACT proteins (1 μg each), representing those that were tested for GST-Us11C binding. Lanes 1 to 4, histidine-tagged PACT proteins Western blotted with anti-histidine antibody; lane 1, wt PACT; lane 2, PACTΔ1; lane 3, PACTΔ1,2; lane 4, PACTΔ3; lane 5, MBP-3 Western blotted with anti-PACT domain 3 antibody. (B to D) Proteins pulled down with glutathione-Sepharose 4B and Western blotted with anti-histidine antibody (B), anti-PACT domain 3 antibody (C), and anti-GST antibody (D). In panels B to D, all lanes except lanes 1 contained GST-Us11C. The additional proteins were as follows: wt PACT, PACTΔ1, PACTΔ1,2, PACTΔ3, and MBP-3 (lanes 1); wt PACT (lanes 2); PACTΔ1 (lanes 3); PACTΔ1,2 (lanes 4); PACTΔ3 (lanes 5); and MBP-3 (lanes 6)
Figure Legend Snippet: Bacterially expressed PACT and PACTΔ3, but not Δ1,2, Δ1, or MBP-3, bind Us11C. GST pulldown assays were used to measure the binding of PACT and PACT mutants to GST-tagged Us11C. One microgram of purified PACT or PACT mutant protein was mixed with 1 μg of purified GST-Us11C in high-salt buffer. Glutathione-Sepharose 4B was used to pull down GST-Us11C, and the proteins interacting with it were analyzed. (A) Purified PACT proteins (1 μg each), representing those that were tested for GST-Us11C binding. Lanes 1 to 4, histidine-tagged PACT proteins Western blotted with anti-histidine antibody; lane 1, wt PACT; lane 2, PACTΔ1; lane 3, PACTΔ1,2; lane 4, PACTΔ3; lane 5, MBP-3 Western blotted with anti-PACT domain 3 antibody. (B to D) Proteins pulled down with glutathione-Sepharose 4B and Western blotted with anti-histidine antibody (B), anti-PACT domain 3 antibody (C), and anti-GST antibody (D). In panels B to D, all lanes except lanes 1 contained GST-Us11C. The additional proteins were as follows: wt PACT, PACTΔ1, PACTΔ1,2, PACTΔ3, and MBP-3 (lanes 1); wt PACT (lanes 2); PACTΔ1 (lanes 3); PACTΔ1,2 (lanes 4); PACTΔ3 (lanes 5); and MBP-3 (lanes 6)

Techniques Used: Binding Assay, Purification, Mutagenesis, Western Blot

8) Product Images from "p120 Catenin-Associated Fer and Fyn Tyrosine Kinases Regulate ?-Catenin Tyr-142 Phosphorylation and ?-Catenin-?-Catenin Interaction"

Article Title: p120 Catenin-Associated Fer and Fyn Tyrosine Kinases Regulate ?-Catenin Tyr-142 Phosphorylation and ?-Catenin-?-Catenin Interaction

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.7.2287-2297.2003

p120 catenin recruits Fer and Fyn kinases to the junctional complex. IEC18 or IEC18 K-ras total cell extracts (300 μg) were immunoprecipitated with anti-p120 catenin (A) or anti-Fyn (B) antibodies, and this was followed by immunoblotting with the indicated MAbs. Lane NI corresponds to the result of an immunoprecipitation performed with an irrelevant antibody. (C) GST-cytoE-cadherin (15 pmol) was incubated or not with full-length p120 catenin (prephosphorylated with pp60 c-src when indicated). Forty micrograms of total extract prepared from IEC18 K-ras transfected with 5 μg of pcDNA3-His-Fer was added. Protein complexes were pelleted down by affinity on glutathione-Sepharose beads, and proteins bound to the complex were analyzed by SDS-PAGE and Western blotting with MAbs against Fyn, the Xpress epitope, Yes, and p120 catenin. The estimated molecular masses of the bands detected with each antibody are indicated as well as the position of immunoglobulin G (IgG) heavy chains.
Figure Legend Snippet: p120 catenin recruits Fer and Fyn kinases to the junctional complex. IEC18 or IEC18 K-ras total cell extracts (300 μg) were immunoprecipitated with anti-p120 catenin (A) or anti-Fyn (B) antibodies, and this was followed by immunoblotting with the indicated MAbs. Lane NI corresponds to the result of an immunoprecipitation performed with an irrelevant antibody. (C) GST-cytoE-cadherin (15 pmol) was incubated or not with full-length p120 catenin (prephosphorylated with pp60 c-src when indicated). Forty micrograms of total extract prepared from IEC18 K-ras transfected with 5 μg of pcDNA3-His-Fer was added. Protein complexes were pelleted down by affinity on glutathione-Sepharose beads, and proteins bound to the complex were analyzed by SDS-PAGE and Western blotting with MAbs against Fyn, the Xpress epitope, Yes, and p120 catenin. The estimated molecular masses of the bands detected with each antibody are indicated as well as the position of immunoglobulin G (IgG) heavy chains.

Techniques Used: Immunoprecipitation, Incubation, Transfection, SDS Page, Western Blot

Fer and Fyn kinases are activated in IEC18 K-ras. (A) IEC18 or IEC18 K-ras cells were transfected with 5 μg of pcDNA3-His-Fer or empty vector as control. After 48 h, cell extracts were prepared and His-tagged Fer kinase was purified. Proteins bound to the nickel-agarose were subjected to SDS-PAGE and Western blotting with anti-Xpress antibody to verify that similar levels of the transgene were expressed. Membrane was stripped and reblotted to analyze the PTyr content of Fer. (B) The activity of purified Fer was determined by adding 0.4 μg of GST-β-catenin under the phosphorylation conditions indicated in Materials and Methods for 1 h at 30°C. Samples were subjected to SDS-PAGE and Western blotting with antibodies against PTyr, β-catenin, and Xpress (to check that similar amounts of β-catenin and Fer were present in the reactions). (C and D) Assays were carried out as in panels A and B except that IEC18 or IEC18 K-ras cells were transfected with 5 μg of pEF-Bos-Fyn and extracts immunoprecipitated with anti-Fyn antibody. Determination of the β-catenin kinase activity present in the immunoprecipitate was performed as in panel B. The position of immunoglobulin G (IgG) heavy chains is indicated.
Figure Legend Snippet: Fer and Fyn kinases are activated in IEC18 K-ras. (A) IEC18 or IEC18 K-ras cells were transfected with 5 μg of pcDNA3-His-Fer or empty vector as control. After 48 h, cell extracts were prepared and His-tagged Fer kinase was purified. Proteins bound to the nickel-agarose were subjected to SDS-PAGE and Western blotting with anti-Xpress antibody to verify that similar levels of the transgene were expressed. Membrane was stripped and reblotted to analyze the PTyr content of Fer. (B) The activity of purified Fer was determined by adding 0.4 μg of GST-β-catenin under the phosphorylation conditions indicated in Materials and Methods for 1 h at 30°C. Samples were subjected to SDS-PAGE and Western blotting with antibodies against PTyr, β-catenin, and Xpress (to check that similar amounts of β-catenin and Fer were present in the reactions). (C and D) Assays were carried out as in panels A and B except that IEC18 or IEC18 K-ras cells were transfected with 5 μg of pEF-Bos-Fyn and extracts immunoprecipitated with anti-Fyn antibody. Determination of the β-catenin kinase activity present in the immunoprecipitate was performed as in panel B. The position of immunoglobulin G (IgG) heavy chains is indicated.

Techniques Used: Transfection, Plasmid Preparation, Purification, SDS Page, Western Blot, Activity Assay, Immunoprecipitation

Yes kinase activates Fer and Fyn. (A) IEC18 cells were transfected with 5 μg of pMIK-Neo-Yes kinase (wild type or the constitutively active form Tyr-535→Phe) or empty vector. After 48 h, cell extracts were prepared and 200 μg of total cell extracts were immunoprecipitated with anti-Fyn antibody. The immune complexes were analyzed with an anti-PTyr MAb or subjected to an in vitro kinase assay with 0.5 μg of GST-β-catenin as exogenous substrate and analyzed by sequential immunoblotting with antibodies against PTyr and Fyn. The position of immunoglobulin G (IgG) heavy chains is indicated. Very little phosphorylation was obtained when a similar assay was performed using GST-β-catenin (Y142F) as substrate (not shown). (B) IEC18 cells were transfected with 5 μg of pcDNA3-His-Fer kinase. After 48 h, cell extracts were prepared and His-tagged Fer kinase was purified by chromatography on Ni 2+ -agarose and eluted with 60 μl of lysis buffer plus 200 mM imidazole. In parallel, 300 μg of IEC18 or IEC18 K-ras total cell extracts was immunoprecipitated with anti-Yes antibody. The immune complexes were mixed with 20 μl of eluted-Fer kinase and incubated 1 h at 30°C in conditions indicated for kinase assays. Supernatants containing Fer were separated from the immunocomplexes spinning 30 s in a microcentrifuge and subjected to a GST-β-catenin kinase assay as mentioned above. The extent of tyrosine phosphorylation was analyzed blotting with an anti-kinase PTyr MAb. As control to verify that similar amounts of Yes were immunoprecipitated, immunocomplexes were also analyzed by Western blotting with anti-Yes antibody. Lane NI corresponds to the β-catenin phosphorylation obtained when the 20 μl of eluted-Fer was incubated with an IEC18 K-ras cell extract immunoprecipitated with an irrelevant antibody. No phosphorylation of β-catenin was observed when Fer kinase was omitted from the reaction or when GST-β-catenin (Y142F) was used as substrate. The molecular masses of 120 and 95 kDa correspond to GST-β-catenin and Fer, respectively.
Figure Legend Snippet: Yes kinase activates Fer and Fyn. (A) IEC18 cells were transfected with 5 μg of pMIK-Neo-Yes kinase (wild type or the constitutively active form Tyr-535→Phe) or empty vector. After 48 h, cell extracts were prepared and 200 μg of total cell extracts were immunoprecipitated with anti-Fyn antibody. The immune complexes were analyzed with an anti-PTyr MAb or subjected to an in vitro kinase assay with 0.5 μg of GST-β-catenin as exogenous substrate and analyzed by sequential immunoblotting with antibodies against PTyr and Fyn. The position of immunoglobulin G (IgG) heavy chains is indicated. Very little phosphorylation was obtained when a similar assay was performed using GST-β-catenin (Y142F) as substrate (not shown). (B) IEC18 cells were transfected with 5 μg of pcDNA3-His-Fer kinase. After 48 h, cell extracts were prepared and His-tagged Fer kinase was purified by chromatography on Ni 2+ -agarose and eluted with 60 μl of lysis buffer plus 200 mM imidazole. In parallel, 300 μg of IEC18 or IEC18 K-ras total cell extracts was immunoprecipitated with anti-Yes antibody. The immune complexes were mixed with 20 μl of eluted-Fer kinase and incubated 1 h at 30°C in conditions indicated for kinase assays. Supernatants containing Fer were separated from the immunocomplexes spinning 30 s in a microcentrifuge and subjected to a GST-β-catenin kinase assay as mentioned above. The extent of tyrosine phosphorylation was analyzed blotting with an anti-kinase PTyr MAb. As control to verify that similar amounts of Yes were immunoprecipitated, immunocomplexes were also analyzed by Western blotting with anti-Yes antibody. Lane NI corresponds to the β-catenin phosphorylation obtained when the 20 μl of eluted-Fer was incubated with an IEC18 K-ras cell extract immunoprecipitated with an irrelevant antibody. No phosphorylation of β-catenin was observed when Fer kinase was omitted from the reaction or when GST-β-catenin (Y142F) was used as substrate. The molecular masses of 120 and 95 kDa correspond to GST-β-catenin and Fer, respectively.

Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, In Vitro, Kinase Assay, Purification, Chromatography, Lysis, Incubation, Western Blot

In IEC18 K-ras β-catenin, Tyr-142 and Tyr-654 are phosphorylated. Phosphorylation of Tyr-142 modulates the α-catenin-β-catenin interaction in vivo. (A) IEC18 K-ras cells were transfected with 5 μg of pcDNA3.1His-β-catenin (wild type or the indicated mutant forms) or empty vector as control. After 48 h, cell extracts were prepared, His-tagged β-catenin was purified by chromatography on nickel-agarose, and the level of phosphorylation was analyzed by Western blotting with PTyr MAb. Membrane was reanalyzed against β-catenin to check that similar levels of expression were obtained in all the cases. Y86F, Y142F, and Y654F correspond to β-catenin mutants Tyr-86→Phe, Tyr-142→Phe, and Tyr-654→Phe, respectively. (B) IEC18 K-ras cells were transfected with 5 μg of pcDNA3.1His-β-catenin (wild type or Tyr-142→Phe). His-tagged β-catenin complexes were analyzed with the indicated MAbs.
Figure Legend Snippet: In IEC18 K-ras β-catenin, Tyr-142 and Tyr-654 are phosphorylated. Phosphorylation of Tyr-142 modulates the α-catenin-β-catenin interaction in vivo. (A) IEC18 K-ras cells were transfected with 5 μg of pcDNA3.1His-β-catenin (wild type or the indicated mutant forms) or empty vector as control. After 48 h, cell extracts were prepared, His-tagged β-catenin was purified by chromatography on nickel-agarose, and the level of phosphorylation was analyzed by Western blotting with PTyr MAb. Membrane was reanalyzed against β-catenin to check that similar levels of expression were obtained in all the cases. Y86F, Y142F, and Y654F correspond to β-catenin mutants Tyr-86→Phe, Tyr-142→Phe, and Tyr-654→Phe, respectively. (B) IEC18 K-ras cells were transfected with 5 μg of pcDNA3.1His-β-catenin (wild type or Tyr-142→Phe). His-tagged β-catenin complexes were analyzed with the indicated MAbs.

Techniques Used: In Vivo, Transfection, Mutagenesis, Plasmid Preparation, Purification, Chromatography, Western Blot, Expressing

9) Product Images from "Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein"

Article Title: Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein

Journal: Scientific Reports

doi: 10.1038/srep22502

Binding of translation initiation factors to 5′ UTRs of various mRNAs in HeLa cell lysates obtained under mTOR inhibition, serum starvation, or hypoxia. ( A ) A scheme of fragments used in experiment. Fragments with indicated 5′ UTRs and a 106 nt sequence of the luciferase mRNA-encoding region were 3′-biotinylated and 5′-capped. ( B , C ) The biotinylated, capped RNA fragments (2 pmol each) were incubated with 150 μl lysates of Torin2-treated, serum starved, exposed to hypoxia or untreated HeLa cells and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and detected using appropriate antibodies.
Figure Legend Snippet: Binding of translation initiation factors to 5′ UTRs of various mRNAs in HeLa cell lysates obtained under mTOR inhibition, serum starvation, or hypoxia. ( A ) A scheme of fragments used in experiment. Fragments with indicated 5′ UTRs and a 106 nt sequence of the luciferase mRNA-encoding region were 3′-biotinylated and 5′-capped. ( B , C ) The biotinylated, capped RNA fragments (2 pmol each) were incubated with 150 μl lysates of Torin2-treated, serum starved, exposed to hypoxia or untreated HeLa cells and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and detected using appropriate antibodies.

Techniques Used: Binding Assay, Inhibition, Sequencing, Luciferase, Incubation, SDS Page

The effect of 4E-BP(4Ala) on translation of reporter mRNAs and on YB-1 synthesis in HeLa cells. ( A ) 10 5 HeLa cells were transfected by 1 or 2 μg pcDNA3-3HA-4E-BP1(4Ala) or 2 μg pcDNA3-HA plasmids, cultivated for 36 h and used for Western-blot analysis with anti-4E-BP1 antibody. ( B ) 4E-BP1(4Ala)-overexpressing or control HeLa cells (10 5 each) were transfected by reporter Firefly luciferase mRNAs with YB-1 mRNA- or globin mRNA 5′ UTR and Renilla luciferase mRNA (as internal control), cultivated for 2 h, harvested and assayed for Firefly and Renilla luciferase ( FLuc and RLuc , respectively). The FLuc / RLuc ratio for the control (untreated cells) was taken as 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. ( C ) 4E-BP1(4Ala)-overexpressing or control HeLa cells (10 6 each) were labeled with [ 35 S]-methionine for 2 h, harvested and lysed. Cell lysates were used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [ 35 S]-labeled proteins were detected by autoradiography. ( D ) Biotinylated, capped luciferase mRNA with YB-1 mRNA 5′ UTR (0.32 pmol) was incubated in 150 μl lysates of 4EBP(4Ala)-overxpressing HeLa cells (2 × 10 5 ) or control HeLa cells (2 × 10 5 ) and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE and analyzed by Western blotting.
Figure Legend Snippet: The effect of 4E-BP(4Ala) on translation of reporter mRNAs and on YB-1 synthesis in HeLa cells. ( A ) 10 5 HeLa cells were transfected by 1 or 2 μg pcDNA3-3HA-4E-BP1(4Ala) or 2 μg pcDNA3-HA plasmids, cultivated for 36 h and used for Western-blot analysis with anti-4E-BP1 antibody. ( B ) 4E-BP1(4Ala)-overexpressing or control HeLa cells (10 5 each) were transfected by reporter Firefly luciferase mRNAs with YB-1 mRNA- or globin mRNA 5′ UTR and Renilla luciferase mRNA (as internal control), cultivated for 2 h, harvested and assayed for Firefly and Renilla luciferase ( FLuc and RLuc , respectively). The FLuc / RLuc ratio for the control (untreated cells) was taken as 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. ( C ) 4E-BP1(4Ala)-overexpressing or control HeLa cells (10 6 each) were labeled with [ 35 S]-methionine for 2 h, harvested and lysed. Cell lysates were used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [ 35 S]-labeled proteins were detected by autoradiography. ( D ) Biotinylated, capped luciferase mRNA with YB-1 mRNA 5′ UTR (0.32 pmol) was incubated in 150 μl lysates of 4EBP(4Ala)-overxpressing HeLa cells (2 × 10 5 ) or control HeLa cells (2 × 10 5 ) and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE and analyzed by Western blotting.

Techniques Used: Transfection, Western Blot, Luciferase, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Incubation, SDS Page

Binding of translation initiation factors to 5′ UTRs of various mRNAs in hippuristanol-treated cells. The biotinylated, capped RNA fragments (2 pmol each) were incubated with 150 μl lysates of hippuristanol-treated or untreated HeLa cells and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and detected using appropriate antibodies.
Figure Legend Snippet: Binding of translation initiation factors to 5′ UTRs of various mRNAs in hippuristanol-treated cells. The biotinylated, capped RNA fragments (2 pmol each) were incubated with 150 μl lysates of hippuristanol-treated or untreated HeLa cells and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and detected using appropriate antibodies.

Techniques Used: Binding Assay, Incubation, SDS Page

The effect of Torin2 on translation of reporter mRNAs in HeLa cells, and the effect of 4E-BP1 on translation of reporter mRNAs in vitro . ( A ) Untreated or Torin2-treated (0.25 μM, 1 h) HeLa cells were transfected by reporter Firefly luciferase mRNAs with indicated 5′ UTRs and Renilla luciferase mRNA (as internal control), cultivated for 2 h, harvested and assayed for Firefly and Renilla luciferase ( FLuc and RLuc , respectively). Absolute values of FLuc or RLuc activities are presented. Errors are 2 standard deviations. ( B ) The same as ( A ) but the FLuc / RLuc ratio is presented. The FLuc / RLuc ratio for the control (untreated cells) was taken as 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. ( C ), 0.1 pmol C + A + reporter Firefly luciferase mRNAs with indicated 5′ UTRs were translated in Krebs extract in the presence of increasing amounts of recombinant 4E-BP1 (0.4, 0.8, and 1.6 pmol) or without it. Reaction mixtures were assayed for Firefly luciferase after 45 min incubation at 30 °C. The FLuc activity without addition of 4E-BP1 was taken to be 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. ( D ) Translation reactions (Krebs extract) with 1.6 pmol 4E-BP1 or without it were analyzed by Western blotting (left panel) or used for analysis of binding of translation initiation factors. The biotinylated, capped luciferase mRNA with β-globin mRNA 5′ UTR (0.1 pmol) was incubated in 10 μl translation reaction mixture (Krebs extract) with or without 1.6 pmol 4E-BP1 and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blotting (right panel).
Figure Legend Snippet: The effect of Torin2 on translation of reporter mRNAs in HeLa cells, and the effect of 4E-BP1 on translation of reporter mRNAs in vitro . ( A ) Untreated or Torin2-treated (0.25 μM, 1 h) HeLa cells were transfected by reporter Firefly luciferase mRNAs with indicated 5′ UTRs and Renilla luciferase mRNA (as internal control), cultivated for 2 h, harvested and assayed for Firefly and Renilla luciferase ( FLuc and RLuc , respectively). Absolute values of FLuc or RLuc activities are presented. Errors are 2 standard deviations. ( B ) The same as ( A ) but the FLuc / RLuc ratio is presented. The FLuc / RLuc ratio for the control (untreated cells) was taken as 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. ( C ), 0.1 pmol C + A + reporter Firefly luciferase mRNAs with indicated 5′ UTRs were translated in Krebs extract in the presence of increasing amounts of recombinant 4E-BP1 (0.4, 0.8, and 1.6 pmol) or without it. Reaction mixtures were assayed for Firefly luciferase after 45 min incubation at 30 °C. The FLuc activity without addition of 4E-BP1 was taken to be 100%. Values are the means of at least three independent experiments. Errors are 2 standard deviations. ( D ) Translation reactions (Krebs extract) with 1.6 pmol 4E-BP1 or without it were analyzed by Western blotting (left panel) or used for analysis of binding of translation initiation factors. The biotinylated, capped luciferase mRNA with β-globin mRNA 5′ UTR (0.1 pmol) was incubated in 10 μl translation reaction mixture (Krebs extract) with or without 1.6 pmol 4E-BP1 and immobilized on Streptavidin-Sepharose. RNA-bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blotting (right panel).

Techniques Used: In Vitro, Transfection, Luciferase, Recombinant, Incubation, Activity Assay, Western Blot, Binding Assay, SDS Page

10) Product Images from "The ?-Arrestin-2 Scaffold Protein Promotes c-Jun N-terminal Kinase-3 Activation by Binding to Its Nonconserved N Terminus *"

Article Title: The ?-Arrestin-2 Scaffold Protein Promotes c-Jun N-terminal Kinase-3 Activation by Binding to Its Nonconserved N Terminus *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M710006200

Mapping residues critical for JNK3 binding to β-arrestin-2. A , 38-amino acid N-terminal extension of JNK3. The positions of the N-terminal deletion mutants are indicated. The residues mutated to Ala are in bold italics . B , alignment of the N-terminal sequences of human JNK3 and CAMK1γ. Conserved residues are boxed . Numbers refer to amino acids. C–E , constructs expressing GST or GST-tagged JNK3 mutants were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the JNK3 deletions and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. F , constructs expressing GFP, GFP-JNK3, and GFP-JNK3(L14A/V16A) were introduced into 293T cells and JNK3-containing complexes isolated using anti-GFP antibodies. The presence of endogenous β-arrestin-2 in the precipitates ( IP ) was detected using an anti-β-arrestin antibody. The expression of the JNK3 proteins and β-arrestin-2 was examined by immunoblotting the lysates with anti-GFP and anti-β-arrestin antibodies, respectively. G , constructs expressing GST or GST-tagged β-arrestin-2 were introduced into COS-7 cells together with an expression vector for Myc-tagged CAMK1γ. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the CAMK1γ present in the pull-down ( PD ) was examined by immunoblot using anti-Myc antibody. Experiments were performed either two or three times and representative immunoblots are shown.
Figure Legend Snippet: Mapping residues critical for JNK3 binding to β-arrestin-2. A , 38-amino acid N-terminal extension of JNK3. The positions of the N-terminal deletion mutants are indicated. The residues mutated to Ala are in bold italics . B , alignment of the N-terminal sequences of human JNK3 and CAMK1γ. Conserved residues are boxed . Numbers refer to amino acids. C–E , constructs expressing GST or GST-tagged JNK3 mutants were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the JNK3 deletions and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. F , constructs expressing GFP, GFP-JNK3, and GFP-JNK3(L14A/V16A) were introduced into 293T cells and JNK3-containing complexes isolated using anti-GFP antibodies. The presence of endogenous β-arrestin-2 in the precipitates ( IP ) was detected using an anti-β-arrestin antibody. The expression of the JNK3 proteins and β-arrestin-2 was examined by immunoblotting the lysates with anti-GFP and anti-β-arrestin antibodies, respectively. G , constructs expressing GST or GST-tagged β-arrestin-2 were introduced into COS-7 cells together with an expression vector for Myc-tagged CAMK1γ. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the CAMK1γ present in the pull-down ( PD ) was examined by immunoblot using anti-Myc antibody. Experiments were performed either two or three times and representative immunoblots are shown.

Techniques Used: Binding Assay, Construct, Expressing, Plasmid Preparation, Isolation, Western Blot

Specificity determinants within β-arrestin-2 for binding to JNK3. A , schematic of the β-arrestin constructs used in the experiments and a summary of their binding to JNK3 (- represents weak or no binding; + represents strong binding). The position of the D-domain like sequence is indicated. B and C , constructs expressing GST-tagged β-arrestin-2 deletion mutants were introduced into COS-7 cells together with expression vectors for HA-JNK3 ( B ) and HA-ASK1 ( C ). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the JNK3 or ASK1 present in the pull-down ( PD ) was examined by immunoblot using anti-HA antibody. The expression of the ASK1, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-HA and anti-GST antibodies, respectively. D and E , constructs expressing the indicated GST-tagged β-arrestin proteins were introduced into COS-7 cells together with an expression vector for HA-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the JNK3 present in the pull-down ( PD ) was examined by immunoblot using anti-HA antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-HA and anti-GST antibodies, respectively. F , sequence alignment of the putative D-domains in β-arrestin-1 and β-arrestin-2. The D-domains are boxed , and the key Ser and Pro residues highlighted . Numbers refer to the amino acid position. G , constructs expressing the indicated FLAG-tagged β-arrestin-2 mutants were introduced into COS-7 cells together with an expression vector for either GST or GST-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin proteins present in the pull-down ( PD ) was examined by immunoblot using the M2 antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. H , constructs expressing the indicated FLAG-tagged β-arrestin proteins were introduced into COS-7 cells together with an expression vector for GST-JNK3. FLAG-β-arrestin-2 complexes were isolated from cell lysates by immunoprecipitation with the M2 antibody ( M2-IP ), and JNK3 present in the precipitates was examined by immunoblot using an anti-GST antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. I , scheme of β-arrestin-2 scaffold assembly. The C terminus of β-arrestin-2 (denoted C ) binds to the extended N terminus of JNK3 (denoted N ), and this binding is controlled by residues within the D-domain of β-arrestin-2 (denoted D ). MKK4 is recruited to the complex via its D-domain (denoted D ) binding to JNK3. ASK1 binds to the N terminus of β-arrestin-2 (denoted N ). Experiments were performed either two or three times, and representative immunoblots are shown.
Figure Legend Snippet: Specificity determinants within β-arrestin-2 for binding to JNK3. A , schematic of the β-arrestin constructs used in the experiments and a summary of their binding to JNK3 (- represents weak or no binding; + represents strong binding). The position of the D-domain like sequence is indicated. B and C , constructs expressing GST-tagged β-arrestin-2 deletion mutants were introduced into COS-7 cells together with expression vectors for HA-JNK3 ( B ) and HA-ASK1 ( C ). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the JNK3 or ASK1 present in the pull-down ( PD ) was examined by immunoblot using anti-HA antibody. The expression of the ASK1, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-HA and anti-GST antibodies, respectively. D and E , constructs expressing the indicated GST-tagged β-arrestin proteins were introduced into COS-7 cells together with an expression vector for HA-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the JNK3 present in the pull-down ( PD ) was examined by immunoblot using anti-HA antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-HA and anti-GST antibodies, respectively. F , sequence alignment of the putative D-domains in β-arrestin-1 and β-arrestin-2. The D-domains are boxed , and the key Ser and Pro residues highlighted . Numbers refer to the amino acid position. G , constructs expressing the indicated FLAG-tagged β-arrestin-2 mutants were introduced into COS-7 cells together with an expression vector for either GST or GST-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin proteins present in the pull-down ( PD ) was examined by immunoblot using the M2 antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. H , constructs expressing the indicated FLAG-tagged β-arrestin proteins were introduced into COS-7 cells together with an expression vector for GST-JNK3. FLAG-β-arrestin-2 complexes were isolated from cell lysates by immunoprecipitation with the M2 antibody ( M2-IP ), and JNK3 present in the precipitates was examined by immunoblot using an anti-GST antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. I , scheme of β-arrestin-2 scaffold assembly. The C terminus of β-arrestin-2 (denoted C ) binds to the extended N terminus of JNK3 (denoted N ), and this binding is controlled by residues within the D-domain of β-arrestin-2 (denoted D ). MKK4 is recruited to the complex via its D-domain (denoted D ) binding to JNK3. ASK1 binds to the N terminus of β-arrestin-2 (denoted N ). Experiments were performed either two or three times, and representative immunoblots are shown.

Techniques Used: Binding Assay, Construct, Sequencing, Expressing, Isolation, Plasmid Preparation, Immunoprecipitation, Western Blot

JNK3 recruits MKK4 to β-arrestin-2. A , constructs expressing HA-ASK1 and FLAG-β-arrestin-2 were introduced into COS-7 cells. In the control an empty vector was transfected in place of the β-arrestin-2 expression vector. β-Arrestin-2 was immunoprecipitated with the M2 antibody ( M2-IP ) and ASK1 present in the precipitates detected with anti-HA antibody. The expression of ASK1 and β-arrestin-2 was examined by immunoblotting the lysates with anti-HA and M2 antibodies, respectively. B and C , constructs expressing GST or GST-MKK4 were introduced into COS-7 cells together with an expression vector for HA-ASK1 ( B ) or HA-JNK3 ( C ). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the ASK1 or JNK3 present in the pull-down ( PD ) was examined by immunoblot using anti-HA antibody. The expression of the MKK4, ASK1, and JNK3 was examined by immunoblotting the lysates with anti-GST and anti-HA antibodies, respectively. D , constructs expressing GST-tagged MKK4 were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2 and either HA-JNK3 or HA-ASK1. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the MKK4, β-arrestin-2, ASK1, and JNK3 was examined by immunoblotting the lysates with anti-GST, M2, and anti-HA antibodies, respectively. E , construct expressing GST-MKK4 was introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2 and the indicated JNK mutants. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the MKK4, β-arrestin-2, and the JNK mutants was examined by immunoblotting the lysates with anti-GST, M2, and anti-HA antibodies, respectively. * indicates nonspecific bands detected by the HA antibody. Experiments were performed either two or three times and representative immunoblots are shown.
Figure Legend Snippet: JNK3 recruits MKK4 to β-arrestin-2. A , constructs expressing HA-ASK1 and FLAG-β-arrestin-2 were introduced into COS-7 cells. In the control an empty vector was transfected in place of the β-arrestin-2 expression vector. β-Arrestin-2 was immunoprecipitated with the M2 antibody ( M2-IP ) and ASK1 present in the precipitates detected with anti-HA antibody. The expression of ASK1 and β-arrestin-2 was examined by immunoblotting the lysates with anti-HA and M2 antibodies, respectively. B and C , constructs expressing GST or GST-MKK4 were introduced into COS-7 cells together with an expression vector for HA-ASK1 ( B ) or HA-JNK3 ( C ). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the ASK1 or JNK3 present in the pull-down ( PD ) was examined by immunoblot using anti-HA antibody. The expression of the MKK4, ASK1, and JNK3 was examined by immunoblotting the lysates with anti-GST and anti-HA antibodies, respectively. D , constructs expressing GST-tagged MKK4 were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2 and either HA-JNK3 or HA-ASK1. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the MKK4, β-arrestin-2, ASK1, and JNK3 was examined by immunoblotting the lysates with anti-GST, M2, and anti-HA antibodies, respectively. E , construct expressing GST-MKK4 was introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2 and the indicated JNK mutants. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the MKK4, β-arrestin-2, and the JNK mutants was examined by immunoblotting the lysates with anti-GST, M2, and anti-HA antibodies, respectively. * indicates nonspecific bands detected by the HA antibody. Experiments were performed either two or three times and representative immunoblots are shown.

Techniques Used: Construct, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Isolation, Western Blot

The extended N terminus of JNK3 binds to β-arrestin-2. A , schematic of the JNK and p38 constructs used in the experiments and a summary of their binding to β-arrestin-2 (- represents no binding; + represents binding). B , constructs expressing GST, GST-JNK3, GST-JNK1, or the chimera GST-J3/J1-A were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and FLAG-β-arrestin-2 (β- Arr2 ) present in the pull-down ( PD ) was examined by immunoblot analysis using the M2 antibody. The expression of the GST-JNK constructs and FLAG-β-arrestin-2 in the lysates was examined by immunoblotting. C and D , constructs expressing GST, GST-JNK3, or GST-JNK3ΔN were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2 ( C ) or FLAG-JIP1 ( D ). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 or JIP1 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the JNK mutants, β-arrestin-2 and JIP1 was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. E and F , constructs expressing GST or the indicated GST-JNK3 mutants were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 present in the pull-downs ( PD ) was examined by immunoblot using M2 antibody. The expression of the JNK mutants and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. Experiments were performed either two or three times and representative immunoblots are shown.
Figure Legend Snippet: The extended N terminus of JNK3 binds to β-arrestin-2. A , schematic of the JNK and p38 constructs used in the experiments and a summary of their binding to β-arrestin-2 (- represents no binding; + represents binding). B , constructs expressing GST, GST-JNK3, GST-JNK1, or the chimera GST-J3/J1-A were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and FLAG-β-arrestin-2 (β- Arr2 ) present in the pull-down ( PD ) was examined by immunoblot analysis using the M2 antibody. The expression of the GST-JNK constructs and FLAG-β-arrestin-2 in the lysates was examined by immunoblotting. C and D , constructs expressing GST, GST-JNK3, or GST-JNK3ΔN were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2 ( C ) or FLAG-JIP1 ( D ). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 or JIP1 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the JNK mutants, β-arrestin-2 and JIP1 was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. E and F , constructs expressing GST or the indicated GST-JNK3 mutants were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 present in the pull-downs ( PD ) was examined by immunoblot using M2 antibody. The expression of the JNK mutants and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. Experiments were performed either two or three times and representative immunoblots are shown.

Techniques Used: Construct, Binding Assay, Expressing, Plasmid Preparation, Isolation, Western Blot

MKK4 is recruited to the β-arrestin-2 scaffold complex via its N-terminal D-domain. A , constructs expressing GST, GST-MKK4, or GST-MKK4 (L44A/L46A) were introduced into COS-7 cells together with an expression vector for HA-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and JNK3 present in the pull-down ( PD ) was examined by immunoblot using anti-HA antibody. The expression of the MKK4 and JNK3 was examined by immunoblotting the lysates with anti-GST and anti-HA antibodies, respectively. B , constructs expressing GST-MKK4 or GST-MKK4 (L44A/L46A) were introduced into COS-7 cells together with expression vectors for HA-JNK3 and FLAG-β-arrestin-2. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the MKK4, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST, anti-HA, and M2 antibodies, respectively. C and D , constructs expressing GST, GST-JNK3, or GST-JNK3(CDmut) were introduced into COS-7 cells together with expression vectors for either FLAG-β-arrestin-2 ( C ) or Myc-tagged MKK4 ( D ). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and β-arrestin-2 and MKK4 present in the pull-down ( PD ) were examined by immunoblot using M2 and anti-Myc antibodies, respectively. The expression of the JNK3, β-arrestin-2, and MKK4 was examined by immunoblotting the lysates with anti-GST, M2, and anti-Myc antibodies, respectively. E , constructs expressing GST-tagged MKK4 were introduced into COS-7 cells together with expression vectors for FLAG-β-arrestin-2 and either HA-JNK3 or HA-JNK3 (CDmut). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the MKK4, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST, anti-HA, and M2 antibodies, respectively. * indicates nonspecific bands detected by the HA antibody. Experiments were performed either two or three times, and representative immunoblots are shown.
Figure Legend Snippet: MKK4 is recruited to the β-arrestin-2 scaffold complex via its N-terminal D-domain. A , constructs expressing GST, GST-MKK4, or GST-MKK4 (L44A/L46A) were introduced into COS-7 cells together with an expression vector for HA-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and JNK3 present in the pull-down ( PD ) was examined by immunoblot using anti-HA antibody. The expression of the MKK4 and JNK3 was examined by immunoblotting the lysates with anti-GST and anti-HA antibodies, respectively. B , constructs expressing GST-MKK4 or GST-MKK4 (L44A/L46A) were introduced into COS-7 cells together with expression vectors for HA-JNK3 and FLAG-β-arrestin-2. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the MKK4, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST, anti-HA, and M2 antibodies, respectively. C and D , constructs expressing GST, GST-JNK3, or GST-JNK3(CDmut) were introduced into COS-7 cells together with expression vectors for either FLAG-β-arrestin-2 ( C ) or Myc-tagged MKK4 ( D ). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and β-arrestin-2 and MKK4 present in the pull-down ( PD ) were examined by immunoblot using M2 and anti-Myc antibodies, respectively. The expression of the JNK3, β-arrestin-2, and MKK4 was examined by immunoblotting the lysates with anti-GST, M2, and anti-Myc antibodies, respectively. E , constructs expressing GST-tagged MKK4 were introduced into COS-7 cells together with expression vectors for FLAG-β-arrestin-2 and either HA-JNK3 or HA-JNK3 (CDmut). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin-2 present in the pull-down ( PD ) was examined by immunoblot using M2 antibody. The expression of the MKK4, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST, anti-HA, and M2 antibodies, respectively. * indicates nonspecific bands detected by the HA antibody. Experiments were performed either two or three times, and representative immunoblots are shown.

Techniques Used: Construct, Expressing, Plasmid Preparation, Isolation, Western Blot

11) Product Images from "A Gα12-specific Binding Domain in AKAP-Lbc and p114RhoGEF"

Article Title: A Gα12-specific Binding Domain in AKAP-Lbc and p114RhoGEF

Journal: Journal of Molecular Signaling

doi: 10.5334/1750-2187-11-3

Conserved residues in AKAP-Lbc and p114RhoGEF participate in Gα12 binding. (A) Interaction of p114RhoGEF charge-reversal mutants with Gα12. The indicated mutants engineered in our GST-fused 257-amino acid region of p114RhoGEF were expressed in E. coli and immobilized on Sepharose. Concentrations of mutant p114RhoGEF proteins were adjusted so that approximately equal concentrations would be compared for ability to co-precipitate constitutively active Gα12 ( 12 QL ). For each sample, 20% of volume was analyzed by Coomassie Blue staining (lower image) to confirm uniform levels of immobilized p114RhoGEF variants. (B) AKAP-Lbc charge-reversal mutants were examined for Gα12 co-precipitation using the same procedure described for p114RhoGEF mutants. (C) For co-precipitations of Gα12, bands were quantified using ImageJ. Precipitate:load values for each mutant were calculated and presented as % of this value for non-mutated p114RhoGEF or AKAP-Lbc regions, which were set at 100%. Graphical data represent two or more independent experiments, with mean ± range shown. (D) Co-precipitation of Gα12 by an immobilized 257-residue p114RhoGEF construct harboring a charge-reversal of the Rgnef-homologous Arg residue ( R760E ). Images are representative of two independent experiments.
Figure Legend Snippet: Conserved residues in AKAP-Lbc and p114RhoGEF participate in Gα12 binding. (A) Interaction of p114RhoGEF charge-reversal mutants with Gα12. The indicated mutants engineered in our GST-fused 257-amino acid region of p114RhoGEF were expressed in E. coli and immobilized on Sepharose. Concentrations of mutant p114RhoGEF proteins were adjusted so that approximately equal concentrations would be compared for ability to co-precipitate constitutively active Gα12 ( 12 QL ). For each sample, 20% of volume was analyzed by Coomassie Blue staining (lower image) to confirm uniform levels of immobilized p114RhoGEF variants. (B) AKAP-Lbc charge-reversal mutants were examined for Gα12 co-precipitation using the same procedure described for p114RhoGEF mutants. (C) For co-precipitations of Gα12, bands were quantified using ImageJ. Precipitate:load values for each mutant were calculated and presented as % of this value for non-mutated p114RhoGEF or AKAP-Lbc regions, which were set at 100%. Graphical data represent two or more independent experiments, with mean ± range shown. (D) Co-precipitation of Gα12 by an immobilized 257-residue p114RhoGEF construct harboring a charge-reversal of the Rgnef-homologous Arg residue ( R760E ). Images are representative of two independent experiments.

Techniques Used: Binding Assay, Mutagenesis, Staining, Construct

A conserved Gα12-binding domain in AKAP-Lbc and p114RhoGEF. (A) Sequence alignment of AKAP-Lbc and p114RhoGEF. Results of Protein BLAST (blastp) analysis are displayed, with black bars indicating identical residues (50/106), white bars indicating non-identical positive matches (28 additional residues), and zero gaps. (B) Interaction of Gα12 with regions of p114RhoGEF and AKAP-Lbc. Co-precipitation experiments were performed as described in Methods, using GST-fusions of the 257-amino acid C-terminus of AKAP-Lbc ( AKAP ), the corresponding 257 residues of p114RhoGEF ( p114 ), amino acids 2-252 of p115RhoGEF containing its RH domain ( RH ), and GST alone. Load samples were set aside prior to addition of Sepharose-bound GST-fusion proteins. Co-precipitations from HEK293 cells transfected with myc-tagged, constitutively activated Gα12 ( 12 QL ) and empty vector were performed in parallel. For each sample, 20% of precipitated material was examined by SDS-PAGE/Coomassie Blue staining to assess levels of GST-fusion proteins, shown in lower panels. (C) Co-precipitations using the closely homologous, 106-amino acid domains within the Gα12-binding, 257-residue regions of AKAP-Lbc ( AKAP ) and p114RhoGEF ( p114 ) are shown. Amino acid lengths of these adducts to GST are indicated as superscripts. Results in (B) and (C) are representative of three or more independent experiments.
Figure Legend Snippet: A conserved Gα12-binding domain in AKAP-Lbc and p114RhoGEF. (A) Sequence alignment of AKAP-Lbc and p114RhoGEF. Results of Protein BLAST (blastp) analysis are displayed, with black bars indicating identical residues (50/106), white bars indicating non-identical positive matches (28 additional residues), and zero gaps. (B) Interaction of Gα12 with regions of p114RhoGEF and AKAP-Lbc. Co-precipitation experiments were performed as described in Methods, using GST-fusions of the 257-amino acid C-terminus of AKAP-Lbc ( AKAP ), the corresponding 257 residues of p114RhoGEF ( p114 ), amino acids 2-252 of p115RhoGEF containing its RH domain ( RH ), and GST alone. Load samples were set aside prior to addition of Sepharose-bound GST-fusion proteins. Co-precipitations from HEK293 cells transfected with myc-tagged, constitutively activated Gα12 ( 12 QL ) and empty vector were performed in parallel. For each sample, 20% of precipitated material was examined by SDS-PAGE/Coomassie Blue staining to assess levels of GST-fusion proteins, shown in lower panels. (C) Co-precipitations using the closely homologous, 106-amino acid domains within the Gα12-binding, 257-residue regions of AKAP-Lbc ( AKAP ) and p114RhoGEF ( p114 ) are shown. Amino acid lengths of these adducts to GST are indicated as superscripts. Results in (B) and (C) are representative of three or more independent experiments.

Techniques Used: Binding Assay, Sequencing, Transfection, Plasmid Preparation, SDS Page, Staining

Gα12 binding to AKAP-Lbc and p114RhoGEF is distinct from RH-RhoGEF interaction. (A, B) Results of protein interaction experiments using mutant forms of myc-tagged, constitutively active Gα12 (Gα12 QL ). For both panels, Sepharose-immobilized AKAP-Lbc and p114RhoGEF constructs utilized in Figure 2B were tested, alongside the immobilized RH domain of LARG ( RH ), for ability to co-precipitate Gα12 variants from HEK293 cell extracts as described in Methods. Load samples were set aside prior to the precipitation step. Representative results are shown. (C) Immunoblot bands for Gα12 mutants were quantified using ImageJ, and for each sample the precipitate:load ratio was calculated and presented as a % of the precipitate:load ratio for non-mutated, constitutively active Gα12. Results shown are from three or more independent experiments, with graphs indicating mean ± standard error of the mean (s.e.m.).
Figure Legend Snippet: Gα12 binding to AKAP-Lbc and p114RhoGEF is distinct from RH-RhoGEF interaction. (A, B) Results of protein interaction experiments using mutant forms of myc-tagged, constitutively active Gα12 (Gα12 QL ). For both panels, Sepharose-immobilized AKAP-Lbc and p114RhoGEF constructs utilized in Figure 2B were tested, alongside the immobilized RH domain of LARG ( RH ), for ability to co-precipitate Gα12 variants from HEK293 cell extracts as described in Methods. Load samples were set aside prior to the precipitation step. Representative results are shown. (C) Immunoblot bands for Gα12 mutants were quantified using ImageJ, and for each sample the precipitate:load ratio was calculated and presented as a % of the precipitate:load ratio for non-mutated, constitutively active Gα12. Results shown are from three or more independent experiments, with graphs indicating mean ± standard error of the mean (s.e.m.).

Techniques Used: Binding Assay, Mutagenesis, Construct

Identification of a Gα12-interacting region in AKAP-Lbc. (A) Sequence alignment of AKAP-Lbc with other Gα12 signaling targets. Results of Expasy SIM alignment using amino acid sequences of human proteins AKAP-Lbc (GenBank: NP_006729), axin-1 (NP_003493), and RGS1 (AAH15510) are shown. Regions of AKAP-Lbc excluding the tandem DH/PH domains were examined for homology to short sequences within RGS-1 and axin. Black vertical dashes indicate identical residues, open dashes indicate residues with similar properties. (B) Binding of Gα12 to an AKAP-Lbc region similar to axin and RGS1. HEK293 cells were transfected with a plasmid encoding myc-tagged, constitutively active Gα12 (GTPase-deficient Q229L mutant), or with pcDNA3.1 vector, and detergent-soluble extracts prepared as described in Methods. For each transfected sample, 5% of diluted extract was set aside as starting material ( Load ), and co-precipitation assays were performed using a Sepharose-bound GST-fusion of a 257-residue region of AKAP-Lbc, or GST alone. After SDS-PAGE and electroblot transfer, nitrocellulose membranes were probed with anti-Gα12 antibody (Santa Cruz Biotechnology; sc-409). Immunoblot images shown are representative of > 5 experiments, with bands visible at the expected size (~45 kDa) for myc-tagged Gα12 and not detected in vector-transfected samples.
Figure Legend Snippet: Identification of a Gα12-interacting region in AKAP-Lbc. (A) Sequence alignment of AKAP-Lbc with other Gα12 signaling targets. Results of Expasy SIM alignment using amino acid sequences of human proteins AKAP-Lbc (GenBank: NP_006729), axin-1 (NP_003493), and RGS1 (AAH15510) are shown. Regions of AKAP-Lbc excluding the tandem DH/PH domains were examined for homology to short sequences within RGS-1 and axin. Black vertical dashes indicate identical residues, open dashes indicate residues with similar properties. (B) Binding of Gα12 to an AKAP-Lbc region similar to axin and RGS1. HEK293 cells were transfected with a plasmid encoding myc-tagged, constitutively active Gα12 (GTPase-deficient Q229L mutant), or with pcDNA3.1 vector, and detergent-soluble extracts prepared as described in Methods. For each transfected sample, 5% of diluted extract was set aside as starting material ( Load ), and co-precipitation assays were performed using a Sepharose-bound GST-fusion of a 257-residue region of AKAP-Lbc, or GST alone. After SDS-PAGE and electroblot transfer, nitrocellulose membranes were probed with anti-Gα12 antibody (Santa Cruz Biotechnology; sc-409). Immunoblot images shown are representative of > 5 experiments, with bands visible at the expected size (~45 kDa) for myc-tagged Gα12 and not detected in vector-transfected samples.

Techniques Used: Sequencing, Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, SDS Page

12) Product Images from "Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity"

Article Title: Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039511

TdT ubiquitylation inhibits its nucleotidyltransferase activity. (A) Ubiquitylation of TdT does not effect its DNA-binding property. His-TdT (180 ng) was incubated in the reaction in vitro ubiquitylation mixture with (lane 3) or without (lane 2) biotinylated ssDNA coupled to streptavidin–agarose. The proteins that bound to DNA were subjected to immunoblot analysis using an anti-TdT antibody. (B) Primer extension assay. GST-TdT bound Glutathione Sepharose 4B was added to the ubiquitylation reaction mixture and then incubated with (lanes 2, 4, and 6) or without (lanes 3, 5, and 7) ATP, for the indicated times. After washing, primer extension was performed in reaction mixture containing the Cy5-labeled 20-mer oligo-dT. After 8% SDS page, the products were visualized on a Typhoon 9200 Gel Imager (GE Healthcare).
Figure Legend Snippet: TdT ubiquitylation inhibits its nucleotidyltransferase activity. (A) Ubiquitylation of TdT does not effect its DNA-binding property. His-TdT (180 ng) was incubated in the reaction in vitro ubiquitylation mixture with (lane 3) or without (lane 2) biotinylated ssDNA coupled to streptavidin–agarose. The proteins that bound to DNA were subjected to immunoblot analysis using an anti-TdT antibody. (B) Primer extension assay. GST-TdT bound Glutathione Sepharose 4B was added to the ubiquitylation reaction mixture and then incubated with (lanes 2, 4, and 6) or without (lanes 3, 5, and 7) ATP, for the indicated times. After washing, primer extension was performed in reaction mixture containing the Cy5-labeled 20-mer oligo-dT. After 8% SDS page, the products were visualized on a Typhoon 9200 Gel Imager (GE Healthcare).

Techniques Used: Activity Assay, Binding Assay, Incubation, In Vitro, Primer Extension Assay, Labeling, SDS Page

The SPA motif in UbcH5a is essential for binding to TdT and for TdT ubiquitylation. (A) Amino acid sequence alignment of E2s. The catalytic cysteine is indicated in boldface; the SPA motif is indicated by the gray background. UbcH5, UbcH6, and UbcH13, which bind to TdT, have a SPA motif in loop 7. (B) Mutation of the SPA motif in UbcH5a to AAA. The UbcH5a mutant (mtUbcH5a) was expressed in E. coli as a His-tagged protein and purified. His-mtUbcH5a (lane 1) and His-wtUbcH5a (lane 2) were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). (C) Reduction of UbcH5a’s binding ability by the mutated SPA motif. His-wtUbcH5a and His-mtUbcH5a were incubated with Glutathione Sepharose 4B-bound GST (lanes 3 and 4, respectively) or GST-TdT (lanes 5 and 6, respectively). Proteins bound to the beads were eluted by boiling with Laemmli buffer and immunoblotted with an anti-His or anti-GST antibody. (D) Formation of thioester adducts of wtUbcH5a and mtUbcH5a expressed in E. coli . The thioester reaction mixture contained Ub, UBE1, and UbcH5a as indicated. The same amount of each UbcH5a was used for CBB staining. After 5 min at RT, reactions were stopped by adding Laemmli buffer with (lanes 3 and 6) or without (lanes 1, 2, 4, and 5) β-mercaptoethanol, and proteins were separated by SDS-PAGE. UbcH5a thioester adducts were detected by immunoblotting using an anti-UbcH5 antibody. (E) TdT is not ubiquitylated by mtUbcH5a. E3-independent (lanes 1 to 6) or Cul3-dependent TdT ubiquitylation by the BPOZ-2/Cul3/Rbx1 complex (lanes 7 to 12) was carried out. TdT was incubated in a ubiquitylation reaction mixture containing E1, ubiquitin, and wtUbcH5a or mtUbcH5a. His-TdT ubiquitylation was detected by immunoblotting with an anti-TdT antibody.
Figure Legend Snippet: The SPA motif in UbcH5a is essential for binding to TdT and for TdT ubiquitylation. (A) Amino acid sequence alignment of E2s. The catalytic cysteine is indicated in boldface; the SPA motif is indicated by the gray background. UbcH5, UbcH6, and UbcH13, which bind to TdT, have a SPA motif in loop 7. (B) Mutation of the SPA motif in UbcH5a to AAA. The UbcH5a mutant (mtUbcH5a) was expressed in E. coli as a His-tagged protein and purified. His-mtUbcH5a (lane 1) and His-wtUbcH5a (lane 2) were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). (C) Reduction of UbcH5a’s binding ability by the mutated SPA motif. His-wtUbcH5a and His-mtUbcH5a were incubated with Glutathione Sepharose 4B-bound GST (lanes 3 and 4, respectively) or GST-TdT (lanes 5 and 6, respectively). Proteins bound to the beads were eluted by boiling with Laemmli buffer and immunoblotted with an anti-His or anti-GST antibody. (D) Formation of thioester adducts of wtUbcH5a and mtUbcH5a expressed in E. coli . The thioester reaction mixture contained Ub, UBE1, and UbcH5a as indicated. The same amount of each UbcH5a was used for CBB staining. After 5 min at RT, reactions were stopped by adding Laemmli buffer with (lanes 3 and 6) or without (lanes 1, 2, 4, and 5) β-mercaptoethanol, and proteins were separated by SDS-PAGE. UbcH5a thioester adducts were detected by immunoblotting using an anti-UbcH5 antibody. (E) TdT is not ubiquitylated by mtUbcH5a. E3-independent (lanes 1 to 6) or Cul3-dependent TdT ubiquitylation by the BPOZ-2/Cul3/Rbx1 complex (lanes 7 to 12) was carried out. TdT was incubated in a ubiquitylation reaction mixture containing E1, ubiquitin, and wtUbcH5a or mtUbcH5a. His-TdT ubiquitylation was detected by immunoblotting with an anti-TdT antibody.

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

UbcH5a or UbcH6 directly binds to TdT and E3-independently ubiquitylates TdT in vitro . (A) Ten E2 enzymes were subjected to SDS-PAGE and stained by CBB. (B) Binding between TdT and E2 enzymes in vitro . Ten purified recombinant His-E2 enzymes were incubated with GST- (lane 2), GST-TdT (lane 3) bound Glutathione Sepharose 4B. Proteins bound to the beads were eluted with Laemmli buffer after boiling. The eluates were subjected to SDS-PAGE and detected by immunoblotting using an anti-His antibody. (C) E3-independent TdT ubiquitylation was carried out by 10 E2 enzymes. The substrate His-TdT was incubated with His-Ub, His-UBE1, and His-tagged E2 as indicated (lanes 2–10). After electrophoresis in a denaturing 7.5% polyacrylamide gel, ubiquitylated TdT was detected using an anti-TdT antibody.
Figure Legend Snippet: UbcH5a or UbcH6 directly binds to TdT and E3-independently ubiquitylates TdT in vitro . (A) Ten E2 enzymes were subjected to SDS-PAGE and stained by CBB. (B) Binding between TdT and E2 enzymes in vitro . Ten purified recombinant His-E2 enzymes were incubated with GST- (lane 2), GST-TdT (lane 3) bound Glutathione Sepharose 4B. Proteins bound to the beads were eluted with Laemmli buffer after boiling. The eluates were subjected to SDS-PAGE and detected by immunoblotting using an anti-His antibody. (C) E3-independent TdT ubiquitylation was carried out by 10 E2 enzymes. The substrate His-TdT was incubated with His-Ub, His-UBE1, and His-tagged E2 as indicated (lanes 2–10). After electrophoresis in a denaturing 7.5% polyacrylamide gel, ubiquitylated TdT was detected using an anti-TdT antibody.

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

TdT directly binds to E2. (A) UbcH5a (E2) binds to TdT in vitro . His-UbcH5a was incubated with GST-bound (lanes 2 and 4) or GST-TdT-bound (lanes 3 and 5) Glutathione Sepharose 4B in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of His-Ub. Proteins bound to the beads were eluted by boiling with Laemmli buffer. The eluates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-His, anti-TdT, or anti-GST antibody. (B) The pol β-like region in TdT binds to UbcH5a. His-TdT deletion mutants were incubated with GST-bound (lane 2) or GST-UbcH5a-bound (lane 3) Glutathione Sepharose 4B. Proteins bound to the beads were eluted with Laemmli buffer after boiling. The eluates were subjected to SDS-PAGE and analyzed by immunoblotting using an anti-His antibody. (C) E3-independent ubiquitylation of either His-TdT (aa 150–509 or wt) was carried out with UBE1, UbcH5a, and Ub. Ubiquitylated proteins were detected by immunoblotting using an anti-TdT antibody. (D) TdT is poly-ubiquitylated through lysine(s) other than Lys48. TdT was ubiquitylated in a reaction mixture without Ub (lane 1) or containing UbcH5a with His-Ub wt (lane 2), His-Ub K48R (lane 3), Me-Ub (lane 4), or lysine-less (K0) Ub (lane 5). Ubiquitylated His-TdT was detected by immunoblotting using an anti-TdT antibody. (E) In vitro ubiquitylation of TdT mutants. E3-independent ubiquitylation was carried out for wild-type (wt) or point-mutated His-TdTs. TdT was ubiquitylated in the reaction mixture containing UBE1, UbcH5a, and Ub. His-TdT ubiquitylation was detected by immunoblotting using an anti-TdT antibody.
Figure Legend Snippet: TdT directly binds to E2. (A) UbcH5a (E2) binds to TdT in vitro . His-UbcH5a was incubated with GST-bound (lanes 2 and 4) or GST-TdT-bound (lanes 3 and 5) Glutathione Sepharose 4B in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of His-Ub. Proteins bound to the beads were eluted by boiling with Laemmli buffer. The eluates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-His, anti-TdT, or anti-GST antibody. (B) The pol β-like region in TdT binds to UbcH5a. His-TdT deletion mutants were incubated with GST-bound (lane 2) or GST-UbcH5a-bound (lane 3) Glutathione Sepharose 4B. Proteins bound to the beads were eluted with Laemmli buffer after boiling. The eluates were subjected to SDS-PAGE and analyzed by immunoblotting using an anti-His antibody. (C) E3-independent ubiquitylation of either His-TdT (aa 150–509 or wt) was carried out with UBE1, UbcH5a, and Ub. Ubiquitylated proteins were detected by immunoblotting using an anti-TdT antibody. (D) TdT is poly-ubiquitylated through lysine(s) other than Lys48. TdT was ubiquitylated in a reaction mixture without Ub (lane 1) or containing UbcH5a with His-Ub wt (lane 2), His-Ub K48R (lane 3), Me-Ub (lane 4), or lysine-less (K0) Ub (lane 5). Ubiquitylated His-TdT was detected by immunoblotting using an anti-TdT antibody. (E) In vitro ubiquitylation of TdT mutants. E3-independent ubiquitylation was carried out for wild-type (wt) or point-mutated His-TdTs. TdT was ubiquitylated in the reaction mixture containing UBE1, UbcH5a, and Ub. His-TdT ubiquitylation was detected by immunoblotting using an anti-TdT antibody.

Techniques Used: In Vitro, Incubation, SDS Page

13) Product Images from "HAX1 regulates E3 ubiquitin ligase activity of cIAPs by promoting their dimerization"

Article Title: HAX1 regulates E3 ubiquitin ligase activity of cIAPs by promoting their dimerization

Journal: Oncotarget

doi:

HAX1 regulates the degradation of NIK (A) MDA-MB-231 cells were transfected with increasing amounts of FLAG-HAX1 (0, 0.8, and 2 μg) for 24 h, and the cell lysates were then examined by immunoblotting using appropriate antibodies. Asterisk indicates nonspecific bands. (B) MDA-MB-231 cells were transfected with siHAX1 or scrambled control for 24 h and the cell lysates were analyzed by immunoblotting using anti-HAX1, anti-NIK, and anti-tubulin antibodies. (C) HEK 293T cells were co-transfected with siHAX1 or scrambled control and FLAG-NIK and HA-ubiquitin. After 24 h, the cell lysates were immunoprecipitated with anti-FLAG M2 antibody-conjugated agarose beads and the resulting complexes were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies (bottom two panel). The expression level was determined using anti-HAX1 and anti-FLAG antibodies (top two panels).
Figure Legend Snippet: HAX1 regulates the degradation of NIK (A) MDA-MB-231 cells were transfected with increasing amounts of FLAG-HAX1 (0, 0.8, and 2 μg) for 24 h, and the cell lysates were then examined by immunoblotting using appropriate antibodies. Asterisk indicates nonspecific bands. (B) MDA-MB-231 cells were transfected with siHAX1 or scrambled control for 24 h and the cell lysates were analyzed by immunoblotting using anti-HAX1, anti-NIK, and anti-tubulin antibodies. (C) HEK 293T cells were co-transfected with siHAX1 or scrambled control and FLAG-NIK and HA-ubiquitin. After 24 h, the cell lysates were immunoprecipitated with anti-FLAG M2 antibody-conjugated agarose beads and the resulting complexes were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies (bottom two panel). The expression level was determined using anti-HAX1 and anti-FLAG antibodies (top two panels).

Techniques Used: Multiple Displacement Amplification, Transfection, Immunoprecipitation, Expressing

HAX1 augments the E3 ligase activity of cIAP2 (A) Purified cIAP2 protein was pre-incubated with increasing amounts of HAX1 proteins and the protein mixture were then incubated with E1, UbcH5b, ubiquitin, and ATP at 37°C for 1 h. The reaction mixtures were analyzed by immunoblotting using an anti-cIAP2 antibody and anti-HAX1 antibody. (B) HEK 293T cells were co-transfected with Myc-cIAP2 and increasing amounts of FLAG-HAX1. After 24 h, the cells were treated with a proteasome inhibitor, MG132 (10 μM), for an additional 7 h. Myc-cIAP2 was precipitated with anti-Myc antibody-conjugated agarose beads and the resulting complexes were analyzed by immunoblotting using an anti-ubiquitin antibody (bottom panel). The expression level of each plasmid was determined using anti-FLAG and anti-Myc antibodies (top two panels). (C) Purified cIAP2 protein was pre-incubated with or without HAX1 protein and the protein mixtures were then incubated with UbcH5b-Ub at 4°C for 1 h. The reaction mixtures were immunoprecipitated with an anti-cIAP2 antibody and immune complexes were then separated by SDS-PAGE. (D) Purified cIAP2 was pre-incubated with or without HAX1 protein and the protein mixtures were then incubated with UbcH5b-Ub at 37°C for the 6 h. The resulting reaction mixtures were subjected to immunoblot using the indicated antibodies to analyze the conversion of the UbcH5b-Ub to free UbcH5b.
Figure Legend Snippet: HAX1 augments the E3 ligase activity of cIAP2 (A) Purified cIAP2 protein was pre-incubated with increasing amounts of HAX1 proteins and the protein mixture were then incubated with E1, UbcH5b, ubiquitin, and ATP at 37°C for 1 h. The reaction mixtures were analyzed by immunoblotting using an anti-cIAP2 antibody and anti-HAX1 antibody. (B) HEK 293T cells were co-transfected with Myc-cIAP2 and increasing amounts of FLAG-HAX1. After 24 h, the cells were treated with a proteasome inhibitor, MG132 (10 μM), for an additional 7 h. Myc-cIAP2 was precipitated with anti-Myc antibody-conjugated agarose beads and the resulting complexes were analyzed by immunoblotting using an anti-ubiquitin antibody (bottom panel). The expression level of each plasmid was determined using anti-FLAG and anti-Myc antibodies (top two panels). (C) Purified cIAP2 protein was pre-incubated with or without HAX1 protein and the protein mixtures were then incubated with UbcH5b-Ub at 4°C for 1 h. The reaction mixtures were immunoprecipitated with an anti-cIAP2 antibody and immune complexes were then separated by SDS-PAGE. (D) Purified cIAP2 was pre-incubated with or without HAX1 protein and the protein mixtures were then incubated with UbcH5b-Ub at 37°C for the 6 h. The resulting reaction mixtures were subjected to immunoblot using the indicated antibodies to analyze the conversion of the UbcH5b-Ub to free UbcH5b.

Techniques Used: Activity Assay, Purification, Incubation, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page

HAX1 facilitates the dimerization of the cIAP2 RING domain (A) Mixtures containing purified His-cIAP2 and GST-BUCR proteins were incubated with increasing amounts of HAX1 or HAX1N proteins and then examined by the His-pull down assay. Briefly, proteins were immunoprecipitated with Ni-NTA agarose resin and the resulting complexes were analyzed by immunoblotting with anti-His and anti-GST antibodies. (B) Quantitation of the results from (A). Data are presented as mean ± SEM (error bars) of three independent experiments. (C) HA-cIAP2 and Myc-cIAP2 proteins were expressed with increasing amounts of FLAG-HAX1 in HEK 293T cells. After 24 h, the cells were treated with or without a proteasome inhibitor, MG132 (10 μM), for an additional 7 h and cell lysates were immunoprecipitated with anti-HA agarose. The resulting complexes were analyzed by immunoblotting using appropriate antibodies. (D) Quantitation of the results from (C). Data are presented as mean ± SEM (error bars) of three independent experiments.
Figure Legend Snippet: HAX1 facilitates the dimerization of the cIAP2 RING domain (A) Mixtures containing purified His-cIAP2 and GST-BUCR proteins were incubated with increasing amounts of HAX1 or HAX1N proteins and then examined by the His-pull down assay. Briefly, proteins were immunoprecipitated with Ni-NTA agarose resin and the resulting complexes were analyzed by immunoblotting with anti-His and anti-GST antibodies. (B) Quantitation of the results from (A). Data are presented as mean ± SEM (error bars) of three independent experiments. (C) HA-cIAP2 and Myc-cIAP2 proteins were expressed with increasing amounts of FLAG-HAX1 in HEK 293T cells. After 24 h, the cells were treated with or without a proteasome inhibitor, MG132 (10 μM), for an additional 7 h and cell lysates were immunoprecipitated with anti-HA agarose. The resulting complexes were analyzed by immunoblotting using appropriate antibodies. (D) Quantitation of the results from (C). Data are presented as mean ± SEM (error bars) of three independent experiments.

Techniques Used: Purification, Incubation, Pull Down Assay, Immunoprecipitation, Quantitation Assay

HAX1 interacts with cIAP2 (A) FLAG-cIAP2 protein was expressed in HEK293T cells and immunoprecipitated with anti-FLAG M2 antibody-conjugated agarose beads. The immune complexes were analyzed by immunoblotting with anti-HAX1 antibody and anti-FLAG antibody. (B) The lysates from MDA-MB-231 cells were incubated with IgG or anti-HAX1 antibody, and the immune complexes were analyzed by immunoblotting with anti-cIAP2 antibody and anti-HAX1 antibody. (C,D) Mixtures containing purified HAX1 protein or/and cIAP2 protein were immunoprecipitated with anti-cIAP2 antibody (C) or anti-HAX1 antibody (D) , and then the immune complexes were analyzed by immunoblotting with anti-cIAP2 antibody and anti-HAX1 antibody. (E) Schematic diagram of various HAX1 deletion mutants. BH; Bcl-2 homology domain, PEST; proline (P), glutamic acid (E), serine (S), and threonine (T)-enriched sequence domain, TM; transmembrane domain. (F) FLAG-cIAP2 protein was expressed with various GFP-HAX1 deletion mutants in HEK 293T cells. FLAG-cIAP2 was precipitated with anti-FALG M2 antibody-conjugated agarose beads and then anti-GFP antibody and anti-FLAG antibody were used to detect HAX-1 mutants and cIAP2. Asterisk indicates nonspecific bands. (G) Schematic diagram of various cIAP2 deletion mutants. (H) After purified FLAG-HAX1 protein was incubated with various His-cIAP2 proteins, His-cIAP2 proteins were subjected to Ni-NTA column to pull down the protein complexes. The resulting complexes were analyzed by immunoblotting with anti-His and anti-FLAG antibodies. Asterisks indicate nonspecific bands.
Figure Legend Snippet: HAX1 interacts with cIAP2 (A) FLAG-cIAP2 protein was expressed in HEK293T cells and immunoprecipitated with anti-FLAG M2 antibody-conjugated agarose beads. The immune complexes were analyzed by immunoblotting with anti-HAX1 antibody and anti-FLAG antibody. (B) The lysates from MDA-MB-231 cells were incubated with IgG or anti-HAX1 antibody, and the immune complexes were analyzed by immunoblotting with anti-cIAP2 antibody and anti-HAX1 antibody. (C,D) Mixtures containing purified HAX1 protein or/and cIAP2 protein were immunoprecipitated with anti-cIAP2 antibody (C) or anti-HAX1 antibody (D) , and then the immune complexes were analyzed by immunoblotting with anti-cIAP2 antibody and anti-HAX1 antibody. (E) Schematic diagram of various HAX1 deletion mutants. BH; Bcl-2 homology domain, PEST; proline (P), glutamic acid (E), serine (S), and threonine (T)-enriched sequence domain, TM; transmembrane domain. (F) FLAG-cIAP2 protein was expressed with various GFP-HAX1 deletion mutants in HEK 293T cells. FLAG-cIAP2 was precipitated with anti-FALG M2 antibody-conjugated agarose beads and then anti-GFP antibody and anti-FLAG antibody were used to detect HAX-1 mutants and cIAP2. Asterisk indicates nonspecific bands. (G) Schematic diagram of various cIAP2 deletion mutants. (H) After purified FLAG-HAX1 protein was incubated with various His-cIAP2 proteins, His-cIAP2 proteins were subjected to Ni-NTA column to pull down the protein complexes. The resulting complexes were analyzed by immunoblotting with anti-His and anti-FLAG antibodies. Asterisks indicate nonspecific bands.

Techniques Used: Immunoprecipitation, Multiple Displacement Amplification, Incubation, Purification, Sequencing

14) Product Images from "Phosphorylation of SKAP by GSK3β ensures chromosome segregation by a temporal inhibition of Kif2b activity"

Article Title: Phosphorylation of SKAP by GSK3β ensures chromosome segregation by a temporal inhibition of Kif2b activity

Journal: Scientific Reports

doi: 10.1038/srep38791

SKAP is phosphorylated by GSK3β. ( a ) Co-immunoprecipitation of exogenous SKAP and GSK3β. Extracts from HEK293T cells were transiently co-transfected with both FLAG-SKAP and GFP (lane 3) or GFP-GSK3β (lane 4). The extracts were incubated with an anti-FLAG mouse antibody agarose beads. The immunoprecipitates were resolved by SDS-PAGE followed by Western blotting analyses. Upper panel, GFP blot; lower panel, FLAG blot. ( b ) SKAP directly binds to kinase GSK3β. Using purified GST-SKAP truncation mutants as the matrices, a GST pull-down assay was performed to bind purified His-tagged GSK3β. Western blotting with anti-His antibody showed a specific interaction (lane 5). ( c ) Representative immunofluorescence images of mitotic cells expressing GFP-GSK3β were fixed and stained with SKAP (red). Scale bars, 10 μm. ( d ) Bacterially recombinant GST-SKAP and GST were incubated with GSK3β kinase purified from Sf9 cells in an in vitro phosphorylation reaction. Samples were separated by SDS-PAGE followed by CBB staining and Western blot with an anti-p-ser/thr antibody. ( e ) Identification of sites on SKAP phosphorylated by GSK3β. Recombinant GST-SKAP was phosphorylated by GSK3β in vitro in the presence of ATP as described in “Materials and Methods”, while the phosphorylation sites on SKAP were identified through Mass Spectrometry. The three phosphorylation sites (Ser232, Ser237, Thr294) identified were shown as red. ( f ) Recombinant GST-SKAP-WT and its mutant GST-SKAP-3A were incubated with GSK3β in the presence or absence of GSK3β inhibitor SB415286 as described in “Materials and Methods”.
Figure Legend Snippet: SKAP is phosphorylated by GSK3β. ( a ) Co-immunoprecipitation of exogenous SKAP and GSK3β. Extracts from HEK293T cells were transiently co-transfected with both FLAG-SKAP and GFP (lane 3) or GFP-GSK3β (lane 4). The extracts were incubated with an anti-FLAG mouse antibody agarose beads. The immunoprecipitates were resolved by SDS-PAGE followed by Western blotting analyses. Upper panel, GFP blot; lower panel, FLAG blot. ( b ) SKAP directly binds to kinase GSK3β. Using purified GST-SKAP truncation mutants as the matrices, a GST pull-down assay was performed to bind purified His-tagged GSK3β. Western blotting with anti-His antibody showed a specific interaction (lane 5). ( c ) Representative immunofluorescence images of mitotic cells expressing GFP-GSK3β were fixed and stained with SKAP (red). Scale bars, 10 μm. ( d ) Bacterially recombinant GST-SKAP and GST were incubated with GSK3β kinase purified from Sf9 cells in an in vitro phosphorylation reaction. Samples were separated by SDS-PAGE followed by CBB staining and Western blot with an anti-p-ser/thr antibody. ( e ) Identification of sites on SKAP phosphorylated by GSK3β. Recombinant GST-SKAP was phosphorylated by GSK3β in vitro in the presence of ATP as described in “Materials and Methods”, while the phosphorylation sites on SKAP were identified through Mass Spectrometry. The three phosphorylation sites (Ser232, Ser237, Thr294) identified were shown as red. ( f ) Recombinant GST-SKAP-WT and its mutant GST-SKAP-3A were incubated with GSK3β in the presence or absence of GSK3β inhibitor SB415286 as described in “Materials and Methods”.

Techniques Used: Immunoprecipitation, Transfection, Incubation, SDS Page, Western Blot, Purification, Pull Down Assay, Immunofluorescence, Expressing, Staining, Recombinant, In Vitro, Mass Spectrometry, Mutagenesis

Phosphorylation of SKAP increases its binding affinity to Kif2b. ( a ) Purified GST-SKAP and SKAP mutants were used as matrices to isolate GFP-Kif2b from HEK293T cell lysates. The isolated proteins were then fractionated by SDS-PAGE followed by CBB staining (lower) and anti-GFP blot (upper). ( b ) Statistical analysis of binding efficiency in ( a ). The ordinate indicates the relative binding ratio of GFP-Kif2b to GST-SKAP and its mutants; the abscissa indicates the corresponding binding assay shown in ( a ). ( c ) Co-immunoprecipitation of exogenous SKAP and Kif2b. 36 h after transfection with FLAG-SKAP and GFP-Kif2b, the HEK293T cells were treated with SB415286 for 30 min (lane 5). The cell extracts were incubated with an anti-FLAG antibody agarose beads, and the immunoprecipitates were resolved by SDS-PAGE followed by Western blots. ( d ) Statistical analysis of binding activity in ( c ). The ordinate indicates the binding ratio of GFP-Kif2b and FLAG-SKAP; the abscissa indicates the corresponding binding assay shown in ( c ). ( e ) Purified GST-SKAP and its mutants were used as matrices to isolate purified MBP-Kif2b deletions. The isolated proteins were fractionated by SDS-PAGE followed by CBB staining (lower) and anti-MBP blot (upper). ( f – g ) Statistical analysis of binding ratio in ( e ). The ordinate indicates the binding ratio of MBP-Kif2b-NT ( f ) or MBP-Kif2b-CT ( g ) to GST-SKAP wild-type and its mutants, while the abscissa indicates the corresponding binding assay shown in ( e ). Note that there is no significant difference between wild-type SKAP and phospho-mimicking SKAP-3E ( p = 0.32) bound to Kif2b-C while binding efficiency of non-phosphorylatable SKAP-3A to Kif2b-C was significantly reduced ( p
Figure Legend Snippet: Phosphorylation of SKAP increases its binding affinity to Kif2b. ( a ) Purified GST-SKAP and SKAP mutants were used as matrices to isolate GFP-Kif2b from HEK293T cell lysates. The isolated proteins were then fractionated by SDS-PAGE followed by CBB staining (lower) and anti-GFP blot (upper). ( b ) Statistical analysis of binding efficiency in ( a ). The ordinate indicates the relative binding ratio of GFP-Kif2b to GST-SKAP and its mutants; the abscissa indicates the corresponding binding assay shown in ( a ). ( c ) Co-immunoprecipitation of exogenous SKAP and Kif2b. 36 h after transfection with FLAG-SKAP and GFP-Kif2b, the HEK293T cells were treated with SB415286 for 30 min (lane 5). The cell extracts were incubated with an anti-FLAG antibody agarose beads, and the immunoprecipitates were resolved by SDS-PAGE followed by Western blots. ( d ) Statistical analysis of binding activity in ( c ). The ordinate indicates the binding ratio of GFP-Kif2b and FLAG-SKAP; the abscissa indicates the corresponding binding assay shown in ( c ). ( e ) Purified GST-SKAP and its mutants were used as matrices to isolate purified MBP-Kif2b deletions. The isolated proteins were fractionated by SDS-PAGE followed by CBB staining (lower) and anti-MBP blot (upper). ( f – g ) Statistical analysis of binding ratio in ( e ). The ordinate indicates the binding ratio of MBP-Kif2b-NT ( f ) or MBP-Kif2b-CT ( g ) to GST-SKAP wild-type and its mutants, while the abscissa indicates the corresponding binding assay shown in ( e ). Note that there is no significant difference between wild-type SKAP and phospho-mimicking SKAP-3E ( p = 0.32) bound to Kif2b-C while binding efficiency of non-phosphorylatable SKAP-3A to Kif2b-C was significantly reduced ( p

Techniques Used: Binding Assay, Purification, Isolation, SDS Page, Staining, Immunoprecipitation, Transfection, Incubation, Western Blot, Activity Assay

Identification of a novel SKAP-Kif2b interaction in mitosis. ( a ) SKAP complexes were immunoprecipitated from HeLa cell extract with anti-SKAP antibody or control IgG. The isolated complexes were separated using a gel fraction followed by Western blotting with corresponding antibodies. HC, heavy chain. ( b ) Co-immunoprecipitation of exogenous SKAP and Kif2b. Extracts from HEK293T cells, transiently co-transfected with FLAG-SKAP and GFP (lane 3) or GFP-Kif2b (lane 4), were incubated with an anti-FLAG mouse antibody agarose beads. The immunoprecipitates were resolved using SDS-PAGE followed by Western blotting analyses. Upper panel, GFP blot; lower panel, FLAG blot. ( c ) SKAP binds specifically to Kif2b. Purified GST-SKAP proteins were used to isolate GFP-Kif2b or GFP-Kif2c from HEK293T cell lysates. The isolated proteins were fractionated by SDS-PAGE followed by CBB staining (lower) and anti-GFP blotting analysis (upper). ( d ) Representative immunofluorescence images of mitotic cells expressed GFP-Kif2b were fixed and stained with SKAP (red). Scale bars, 10 μm.
Figure Legend Snippet: Identification of a novel SKAP-Kif2b interaction in mitosis. ( a ) SKAP complexes were immunoprecipitated from HeLa cell extract with anti-SKAP antibody or control IgG. The isolated complexes were separated using a gel fraction followed by Western blotting with corresponding antibodies. HC, heavy chain. ( b ) Co-immunoprecipitation of exogenous SKAP and Kif2b. Extracts from HEK293T cells, transiently co-transfected with FLAG-SKAP and GFP (lane 3) or GFP-Kif2b (lane 4), were incubated with an anti-FLAG mouse antibody agarose beads. The immunoprecipitates were resolved using SDS-PAGE followed by Western blotting analyses. Upper panel, GFP blot; lower panel, FLAG blot. ( c ) SKAP binds specifically to Kif2b. Purified GST-SKAP proteins were used to isolate GFP-Kif2b or GFP-Kif2c from HEK293T cell lysates. The isolated proteins were fractionated by SDS-PAGE followed by CBB staining (lower) and anti-GFP blotting analysis (upper). ( d ) Representative immunofluorescence images of mitotic cells expressed GFP-Kif2b were fixed and stained with SKAP (red). Scale bars, 10 μm.

Techniques Used: Immunoprecipitation, Isolation, Western Blot, Transfection, Incubation, SDS Page, Purification, Staining, Immunofluorescence

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

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

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200208120

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

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

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

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

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

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

16) Product Images from "Functionalized Nano-adsorbent for Affinity Separation of Proteins"

Article Title: Functionalized Nano-adsorbent for Affinity Separation of Proteins

Journal: Nanoscale Research Letters

doi: 10.1186/s11671-018-2531-4

SDS-PAGE analysis of the purified recombinant GPX3, OST1, and ABI2 proteins. Lanes 1, 4, and 7, E. coli lysate; lanes 2, 5, and 8, the proteins eluted from commercial Glutathione Sepharose 4B (GE Healthcare, USA); lanes 3, 6, and 9, the proteins eluted from SnO 2 /SiO 2 -GSH NSs; lane 10, the marker; lanes 11 and 13, GPX3 obtained after the GST tag is cut off from Glutathione Sepharose 4B bound GST-GPX3 and SnO 2 /SiO 2 -GSH NSs bound GST-tagged GPX3; lanes 12 and 14, GST tag eluted from Glutathione Sepharose 4B and SnO 2 /SiO 2 -GSH NSs
Figure Legend Snippet: SDS-PAGE analysis of the purified recombinant GPX3, OST1, and ABI2 proteins. Lanes 1, 4, and 7, E. coli lysate; lanes 2, 5, and 8, the proteins eluted from commercial Glutathione Sepharose 4B (GE Healthcare, USA); lanes 3, 6, and 9, the proteins eluted from SnO 2 /SiO 2 -GSH NSs; lane 10, the marker; lanes 11 and 13, GPX3 obtained after the GST tag is cut off from Glutathione Sepharose 4B bound GST-GPX3 and SnO 2 /SiO 2 -GSH NSs bound GST-tagged GPX3; lanes 12 and 14, GST tag eluted from Glutathione Sepharose 4B and SnO 2 /SiO 2 -GSH NSs

Techniques Used: SDS Page, Purification, Recombinant, Marker

a In vitro assays of GPX3 redox state: lanes 1 and 3 (the oxidized GPX3) and 2 and 4 (the reduced GPX3) refer to GPX3 obtained after the GST tag is cut off from Glutathione Sepharose 4B and SnO 2 /SiO 2 -GSH bound GST-tagged GPX3, respectively; lane 5, marker. b Assays of peroxidase activity of GPX3
Figure Legend Snippet: a In vitro assays of GPX3 redox state: lanes 1 and 3 (the oxidized GPX3) and 2 and 4 (the reduced GPX3) refer to GPX3 obtained after the GST tag is cut off from Glutathione Sepharose 4B and SnO 2 /SiO 2 -GSH bound GST-tagged GPX3, respectively; lane 5, marker. b Assays of peroxidase activity of GPX3

Techniques Used: In Vitro, Marker, Activity Assay

SDS-PAGE analysis of purified GST-tagged proteins separated by SnO 2 /SiO 2 -GSH NSs. a Lane 1, marker; lane 2, E. coli lysate; lanes 3–6 refer to the fractions washed off from the SnO 2 /SiO 2 -GSH NSs with different concentrations of GSH solution (lane 1, 10 mmol/L; lane 2, 20 mmol/L; lane 3, 50 mmol/L; lane 4, 100 mmol/L). b Lane 1, marker; lane 2, E. coli lysate; lane 3, 1st separation; lane 4, 2nd separation; lane 5, 3rd separation; and lane 6, the fractions washed off from the Glutathione Sepharose 4B
Figure Legend Snippet: SDS-PAGE analysis of purified GST-tagged proteins separated by SnO 2 /SiO 2 -GSH NSs. a Lane 1, marker; lane 2, E. coli lysate; lanes 3–6 refer to the fractions washed off from the SnO 2 /SiO 2 -GSH NSs with different concentrations of GSH solution (lane 1, 10 mmol/L; lane 2, 20 mmol/L; lane 3, 50 mmol/L; lane 4, 100 mmol/L). b Lane 1, marker; lane 2, E. coli lysate; lane 3, 1st separation; lane 4, 2nd separation; lane 5, 3rd separation; and lane 6, the fractions washed off from the Glutathione Sepharose 4B

Techniques Used: SDS Page, Purification, Marker

17) Product Images from "Functionalized Nano-adsorbent for Affinity Separation of Proteins"

Article Title: Functionalized Nano-adsorbent for Affinity Separation of Proteins

Journal: Nanoscale Research Letters

doi: 10.1186/s11671-018-2531-4

SDS-PAGE analysis of the purified recombinant GPX3, OST1, and ABI2 proteins. Lanes 1, 4, and 7, E. coli lysate; lanes 2, 5, and 8, the proteins eluted from commercial Glutathione Sepharose 4B (GE Healthcare, USA); lanes 3, 6, and 9, the proteins eluted from SnO 2 /SiO 2 -GSH NSs; lane 10, the marker; lanes 11 and 13, GPX3 obtained after the GST tag is cut off from Glutathione Sepharose 4B bound GST-GPX3 and SnO 2 /SiO 2 -GSH NSs bound GST-tagged GPX3; lanes 12 and 14, GST tag eluted from Glutathione Sepharose 4B and SnO 2 /SiO 2 -GSH NSs
Figure Legend Snippet: SDS-PAGE analysis of the purified recombinant GPX3, OST1, and ABI2 proteins. Lanes 1, 4, and 7, E. coli lysate; lanes 2, 5, and 8, the proteins eluted from commercial Glutathione Sepharose 4B (GE Healthcare, USA); lanes 3, 6, and 9, the proteins eluted from SnO 2 /SiO 2 -GSH NSs; lane 10, the marker; lanes 11 and 13, GPX3 obtained after the GST tag is cut off from Glutathione Sepharose 4B bound GST-GPX3 and SnO 2 /SiO 2 -GSH NSs bound GST-tagged GPX3; lanes 12 and 14, GST tag eluted from Glutathione Sepharose 4B and SnO 2 /SiO 2 -GSH NSs

Techniques Used: SDS Page, Purification, Recombinant, Marker

a In vitro assays of GPX3 redox state: lanes 1 and 3 (the oxidized GPX3) and 2 and 4 (the reduced GPX3) refer to GPX3 obtained after the GST tag is cut off from Glutathione Sepharose 4B and SnO 2 /SiO 2 -GSH bound GST-tagged GPX3, respectively; lane 5, marker. b Assays of peroxidase activity of GPX3
Figure Legend Snippet: a In vitro assays of GPX3 redox state: lanes 1 and 3 (the oxidized GPX3) and 2 and 4 (the reduced GPX3) refer to GPX3 obtained after the GST tag is cut off from Glutathione Sepharose 4B and SnO 2 /SiO 2 -GSH bound GST-tagged GPX3, respectively; lane 5, marker. b Assays of peroxidase activity of GPX3

Techniques Used: In Vitro, Marker, Activity Assay

SDS-PAGE analysis of purified GST-tagged proteins separated by SnO 2 /SiO 2 -GSH NSs. a Lane 1, marker; lane 2, E. coli lysate; lanes 3–6 refer to the fractions washed off from the SnO 2 /SiO 2 -GSH NSs with different concentrations of GSH solution (lane 1, 10 mmol/L; lane 2, 20 mmol/L; lane 3, 50 mmol/L; lane 4, 100 mmol/L). b Lane 1, marker; lane 2, E. coli lysate; lane 3, 1st separation; lane 4, 2nd separation; lane 5, 3rd separation; and lane 6, the fractions washed off from the Glutathione Sepharose 4B
Figure Legend Snippet: SDS-PAGE analysis of purified GST-tagged proteins separated by SnO 2 /SiO 2 -GSH NSs. a Lane 1, marker; lane 2, E. coli lysate; lanes 3–6 refer to the fractions washed off from the SnO 2 /SiO 2 -GSH NSs with different concentrations of GSH solution (lane 1, 10 mmol/L; lane 2, 20 mmol/L; lane 3, 50 mmol/L; lane 4, 100 mmol/L). b Lane 1, marker; lane 2, E. coli lysate; lane 3, 1st separation; lane 4, 2nd separation; lane 5, 3rd separation; and lane 6, the fractions washed off from the Glutathione Sepharose 4B

Techniques Used: SDS Page, Purification, Marker

18) Product Images from "Human DDX3 Interacts with the HIV-1 Tat Protein to Facilitate Viral mRNA Translation"

Article Title: Human DDX3 Interacts with the HIV-1 Tat Protein to Facilitate Viral mRNA Translation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068665

DDX3 interacts with HIV-1 Tat in vitro and in vivo . A . His-tagged recombinant HIV-1 Tat protein was incubated with GST or GST-DDX3. After GST pull-down, bound proteins were analyzed by immunoblotting with anti-HIV-1 Tat antibody (upper panel). The GST-fusion proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (lower panel). B . The experiment was essentially similar to panel A, except that His-tagged recombinant HIV-1 Rev protein was used in the GST pull-down assay. Bound proteins were analyzed by immunoblotting with anti-HIV-1 Rev antibody (upper panel). C . The experiment was essentially similar to panel A. The assay used recombinant GST, GST-DDX3 (full-length; FL) or GST-DDX3 fragments (amino acids 1-226, 227-535 and 536-661) as bait to pull down His-tagged recombinant HIV-1 Tat protein. Bound proteins were analyzed by immunoblotting with anti-HIV-1 Tat antibody (upper panel). The GST-fusion proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (lower panel). D . FLAG-tagged DDX3 and HIV-1 Tat proteins were transiently co-expressed in HEK293 cells for 48 h. Immunoprecipitation was performed using anti-FLAG M2 agarose. Precipitated proteins were subjected to immunoblotting with anti-HIV-1 Tat antibody (upper panel) or anti-DDX3 antibody (lower panel). Ig H represents the immunoglobulin heavy chain. E . HIV-1 Tat protein was transiently expressed in HEK293 cells for 48 h. Immunoprecipitation was performed using anti-HIV-1 Tat antibody bound to protein A sepharose beads. Precipitated proteins were subjected to immunoblotting with anti-DDX3 antibody (upper panel) or anti-HIV-1 Tat antibody (lower panel).
Figure Legend Snippet: DDX3 interacts with HIV-1 Tat in vitro and in vivo . A . His-tagged recombinant HIV-1 Tat protein was incubated with GST or GST-DDX3. After GST pull-down, bound proteins were analyzed by immunoblotting with anti-HIV-1 Tat antibody (upper panel). The GST-fusion proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (lower panel). B . The experiment was essentially similar to panel A, except that His-tagged recombinant HIV-1 Rev protein was used in the GST pull-down assay. Bound proteins were analyzed by immunoblotting with anti-HIV-1 Rev antibody (upper panel). C . The experiment was essentially similar to panel A. The assay used recombinant GST, GST-DDX3 (full-length; FL) or GST-DDX3 fragments (amino acids 1-226, 227-535 and 536-661) as bait to pull down His-tagged recombinant HIV-1 Tat protein. Bound proteins were analyzed by immunoblotting with anti-HIV-1 Tat antibody (upper panel). The GST-fusion proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (lower panel). D . FLAG-tagged DDX3 and HIV-1 Tat proteins were transiently co-expressed in HEK293 cells for 48 h. Immunoprecipitation was performed using anti-FLAG M2 agarose. Precipitated proteins were subjected to immunoblotting with anti-HIV-1 Tat antibody (upper panel) or anti-DDX3 antibody (lower panel). Ig H represents the immunoglobulin heavy chain. E . HIV-1 Tat protein was transiently expressed in HEK293 cells for 48 h. Immunoprecipitation was performed using anti-HIV-1 Tat antibody bound to protein A sepharose beads. Precipitated proteins were subjected to immunoblotting with anti-DDX3 antibody (upper panel) or anti-HIV-1 Tat antibody (lower panel).

Techniques Used: In Vitro, In Vivo, Recombinant, Incubation, SDS Page, Staining, Pull Down Assay, Immunoprecipitation

Translation of HIV-1 mRNAs is impaired in DDX3-depleted cells. HeLa cells were transfected with empty pSilencer 1.0-U6 vector (mock) or the pSilencer 1.0-U6 vector expressing sh-DDX3#2 (DDX3-KD). After 72 hours, cells were re-transfected with the proviral DNA pHXB2gpt plasmid and harvested at 12 h post-transfection. A . Immunoblotting was performed using anti-DDX3 and anti-α-tubulin antibodies to show the knockdown efficiency of DDX3 in HeLa cells . B . Cytoplasmic extracts prepared from mock-transfected (mock) or DDX3-depleted (DDX3-KD) HeLa cells were subjected to 15-40% sucrose gradient sedimentation. RNA extracted from gradient fractions was analyzed by conventional RT-PCR using specific primers for HIV-1 Tat and Rev mRNAs (upper two panels). The housekeeping gene β-actin mRNA, whose translation is not significantly affected by DDX3 knockdown, served as a negative control (the 3 rd panel). The translational efficiency of each mRNA was calculated as the ratio of polysome-associated mRNAs (fractions 7-11) to total mRNA (all fractions). The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (lower panel). C . RNA extracted from the cytoplasmic (Cyto.) and nuclear (Nu.) fractions of mock-transfected (mock) and DDX3-depleted (DDX3-KD) HeLa cells was analyzed by conventional RT-PCR using specific primers for HIV-1 Tat , HIV-1 Rev , and β-actin mRNAs (upper three panels). The subcellular fractions were also subjected to immunoblotting using anti-lamin A/C and anti-α-tubulin (lower two panels).
Figure Legend Snippet: Translation of HIV-1 mRNAs is impaired in DDX3-depleted cells. HeLa cells were transfected with empty pSilencer 1.0-U6 vector (mock) or the pSilencer 1.0-U6 vector expressing sh-DDX3#2 (DDX3-KD). After 72 hours, cells were re-transfected with the proviral DNA pHXB2gpt plasmid and harvested at 12 h post-transfection. A . Immunoblotting was performed using anti-DDX3 and anti-α-tubulin antibodies to show the knockdown efficiency of DDX3 in HeLa cells . B . Cytoplasmic extracts prepared from mock-transfected (mock) or DDX3-depleted (DDX3-KD) HeLa cells were subjected to 15-40% sucrose gradient sedimentation. RNA extracted from gradient fractions was analyzed by conventional RT-PCR using specific primers for HIV-1 Tat and Rev mRNAs (upper two panels). The housekeeping gene β-actin mRNA, whose translation is not significantly affected by DDX3 knockdown, served as a negative control (the 3 rd panel). The translational efficiency of each mRNA was calculated as the ratio of polysome-associated mRNAs (fractions 7-11) to total mRNA (all fractions). The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (lower panel). C . RNA extracted from the cytoplasmic (Cyto.) and nuclear (Nu.) fractions of mock-transfected (mock) and DDX3-depleted (DDX3-KD) HeLa cells was analyzed by conventional RT-PCR using specific primers for HIV-1 Tat , HIV-1 Rev , and β-actin mRNAs (upper three panels). The subcellular fractions were also subjected to immunoblotting using anti-lamin A/C and anti-α-tubulin (lower two panels).

Techniques Used: Transfection, Plasmid Preparation, Expressing, Sedimentation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Agarose Gel Electrophoresis, Staining

HIV-1 Tat is associated with translating mRNAs and facilitates translation of reporter mRNAs containing the HIV-1 5’ UTR. A . HIV-1 Tat protein was transiently co-expressed with the Fluc reporter mRNA containing the 5’ UTR of HIV-1 mRNAs (HIV-5’ UTR) in HEK293 cells for 24 h. Cytoplasmic extracts were subjected to 15-40% sucrose gradient sedimentation. Proteins and RNAs were recovered from 23 fractions for analysis. Immunoblotting analysis of gradient fractions was performed using antibodies against DDX3, eIF4A1, eIF2α, eIF4E and HIV-1 Tat. The association of HIV-1 Tat with translation initiation complexes and polysomes was also detected in the absence of the HIV-1 5’ UTR reporter (w/o HIV-5’ UTR) and in DDX3-depleted (DDX3-KD) HEK293 cells. The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (lower panel). B . The in vitro translation assay was performed using in vitro -transcribed HIV-1 5’ UTR-containing Fluc mRNA and control Rluc mRNA as templates in HeLa cell lysate supplemented with different amounts of recombinant HIV-1 Tat. The graph shows the relative Fluc/Rluc activities of each reaction relative to that of the corresponding reaction without addition of HIV-1 Tat. All data are shown as mean (± SEM) from at least three independent experiments. C . The experiment was essentially similar to panel B, except that Rluc mRNA harbored the HIV-1 5’ UTR while the non-modefied Fluc mRNA served as a control. The graph shows the relative Rluc/Fluc activities of each reaction relative to that of the corresponding reaction without addition of HIV-1 Tat. All data are shown as mean (± SEM) from at least three independent experiments.
Figure Legend Snippet: HIV-1 Tat is associated with translating mRNAs and facilitates translation of reporter mRNAs containing the HIV-1 5’ UTR. A . HIV-1 Tat protein was transiently co-expressed with the Fluc reporter mRNA containing the 5’ UTR of HIV-1 mRNAs (HIV-5’ UTR) in HEK293 cells for 24 h. Cytoplasmic extracts were subjected to 15-40% sucrose gradient sedimentation. Proteins and RNAs were recovered from 23 fractions for analysis. Immunoblotting analysis of gradient fractions was performed using antibodies against DDX3, eIF4A1, eIF2α, eIF4E and HIV-1 Tat. The association of HIV-1 Tat with translation initiation complexes and polysomes was also detected in the absence of the HIV-1 5’ UTR reporter (w/o HIV-5’ UTR) and in DDX3-depleted (DDX3-KD) HEK293 cells. The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (lower panel). B . The in vitro translation assay was performed using in vitro -transcribed HIV-1 5’ UTR-containing Fluc mRNA and control Rluc mRNA as templates in HeLa cell lysate supplemented with different amounts of recombinant HIV-1 Tat. The graph shows the relative Fluc/Rluc activities of each reaction relative to that of the corresponding reaction without addition of HIV-1 Tat. All data are shown as mean (± SEM) from at least three independent experiments. C . The experiment was essentially similar to panel B, except that Rluc mRNA harbored the HIV-1 5’ UTR while the non-modefied Fluc mRNA served as a control. The graph shows the relative Rluc/Fluc activities of each reaction relative to that of the corresponding reaction without addition of HIV-1 Tat. All data are shown as mean (± SEM) from at least three independent experiments.

Techniques Used: Sedimentation, Agarose Gel Electrophoresis, Staining, In Vitro, Recombinant

19) Product Images from "Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing"

Article Title: Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007094

LRR5 stabilized afadin- InlP interaction. (A) Loading control of InlP-afadin binding pull-down experiments with LRR mutants by Coomassie blue staining. GST protein alone (-), InlPΔLRR5-GST fusion protein (ΔLRR5), InlPΔLRR7-GST fusion protein (ΔLRR7), or InlP-GST (InlP) bound to glutathione-Sepharose resin were used as bait for pull-down experiments with protein extracts from MDCK cell line. Data shown are Coomassie staining, and the most abundant band in each lane represents the bait. (B-C) GST fusion proteins or GST protein alone (-) bound to glutathione-sepharose resin were incubated overnight with protein extracts from MDCK cells, and elution fractions (EF) were analyzed by Western blot with anti-afadin antibodies. The following GST fusion proteins were used: GST-InlP (InlP) and GST-InlP with deletions in LRR5 (ΔLRR5) or LRR7 (ΔLRR7). Actin: loading control. Undiluted elution fractions were analyzed with the exception of the elution fractions marked in panel C that were diluted 5-fold. Actin: loading control. (D) Scatter plot showing the logarithm of the ratio of intensity of InlP-GST bound proteins to the intensity of ΔLRR5-GST bound proteins versus total intensity of InlP-GST bound proteins. The intensities (A.U.) of the proteins were identified through mass spectrometry using InlP-GST or InlPΔLRR5-GST as baits to identify host binding partners in the MDCK cell culture extracts. Blue diamonds show all the proteins apart from afadin, which is indicated as orange square. Using this metric, afadin is 2.7 standard deviations away from the mean for this group.
Figure Legend Snippet: LRR5 stabilized afadin- InlP interaction. (A) Loading control of InlP-afadin binding pull-down experiments with LRR mutants by Coomassie blue staining. GST protein alone (-), InlPΔLRR5-GST fusion protein (ΔLRR5), InlPΔLRR7-GST fusion protein (ΔLRR7), or InlP-GST (InlP) bound to glutathione-Sepharose resin were used as bait for pull-down experiments with protein extracts from MDCK cell line. Data shown are Coomassie staining, and the most abundant band in each lane represents the bait. (B-C) GST fusion proteins or GST protein alone (-) bound to glutathione-sepharose resin were incubated overnight with protein extracts from MDCK cells, and elution fractions (EF) were analyzed by Western blot with anti-afadin antibodies. The following GST fusion proteins were used: GST-InlP (InlP) and GST-InlP with deletions in LRR5 (ΔLRR5) or LRR7 (ΔLRR7). Actin: loading control. Undiluted elution fractions were analyzed with the exception of the elution fractions marked in panel C that were diluted 5-fold. Actin: loading control. (D) Scatter plot showing the logarithm of the ratio of intensity of InlP-GST bound proteins to the intensity of ΔLRR5-GST bound proteins versus total intensity of InlP-GST bound proteins. The intensities (A.U.) of the proteins were identified through mass spectrometry using InlP-GST or InlPΔLRR5-GST as baits to identify host binding partners in the MDCK cell culture extracts. Blue diamonds show all the proteins apart from afadin, which is indicated as orange square. Using this metric, afadin is 2.7 standard deviations away from the mean for this group.

Techniques Used: Binding Assay, Staining, Incubation, Western Blot, Mass Spectrometry, Cell Culture

Distinct features of InlP as compared to Lmo2027. (A) Pairwise sequence alignment of InlP and Lmo2027 with conserved amino acids highlighted in red. The LRRs and calcium binding site on InlP are noted. Arrows signify ß-sheets and coils correspond to α-helices. (B) Loading control of pull down experiments on Lmo2027 by Coomassie blue staining. GST protein alone (-), Lmo2027-GST fusion protein (2027) or InlP-GST fusion protein (InlP) bound to glutathione-Sepharose resin used as bait for pull-down experiments with protein extracts from MDCK cells. The elution fraction from pull-down samples using InlP-GST (InlP) as bait were sequentially diluted 5-fold before SDS analysis; undiluted elution fractions were analyzed from pull-down samples using GST (-) or Lmo2027 (2027). Data shown are Coomassie staining and the most abundant band in each lane represents the bait. (C) GST fusion proteins or GST protein alone (-) bound to glutathione-Sepharose resin were incubated overnight with protein extracts from MDCK cells, and elution fractions were analyzed by Western blot with anti-afadin antibodies. The following GST fusion proteins were used: GST-Lmo2027 (Lmo2027) and GST-InlP (InlP). U ndiluted elution fractions were analyzed with the exception of the elution fractions marked by black triangles: elution fractions from the experiments using GST-InlP as bait were sequentially diluted by 5-fold before western blot analysis. Actin: loading control. (D) Crystal structure of InlP with schematic depiction of domain layout below. (E) Crystal structure of Lmo2027 with color-coded domains, like in (D).
Figure Legend Snippet: Distinct features of InlP as compared to Lmo2027. (A) Pairwise sequence alignment of InlP and Lmo2027 with conserved amino acids highlighted in red. The LRRs and calcium binding site on InlP are noted. Arrows signify ß-sheets and coils correspond to α-helices. (B) Loading control of pull down experiments on Lmo2027 by Coomassie blue staining. GST protein alone (-), Lmo2027-GST fusion protein (2027) or InlP-GST fusion protein (InlP) bound to glutathione-Sepharose resin used as bait for pull-down experiments with protein extracts from MDCK cells. The elution fraction from pull-down samples using InlP-GST (InlP) as bait were sequentially diluted 5-fold before SDS analysis; undiluted elution fractions were analyzed from pull-down samples using GST (-) or Lmo2027 (2027). Data shown are Coomassie staining and the most abundant band in each lane represents the bait. (C) GST fusion proteins or GST protein alone (-) bound to glutathione-Sepharose resin were incubated overnight with protein extracts from MDCK cells, and elution fractions were analyzed by Western blot with anti-afadin antibodies. The following GST fusion proteins were used: GST-Lmo2027 (Lmo2027) and GST-InlP (InlP). U ndiluted elution fractions were analyzed with the exception of the elution fractions marked by black triangles: elution fractions from the experiments using GST-InlP as bait were sequentially diluted by 5-fold before western blot analysis. Actin: loading control. (D) Crystal structure of InlP with schematic depiction of domain layout below. (E) Crystal structure of Lmo2027 with color-coded domains, like in (D).

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

Bacterial effector InlP binds host adaptor protein afadin. (A-B) InlP-GST fusion protein (InlP) or GST protein alone (-) bound to glutathione-Sepharose resin were incubated overnight with protein extracts from (A) MDCK or MDCK AF-6 -/- cells or (B) human placenta. Input (IN) and elution fractions (EF) were analyzed by Western blot with anti-afadin antibodies. Actin: loading control. (C-E) Immunofluorescence of human placental villi stained for afadin (red) and DAPI (blue). In panel C the white bar = 10 μm, the asterisk labels a portion of the stroma, and the arrow points to some of the CTBs. White rectangles in panel C indicate the locations of zoomed insets shown in panels D-E.
Figure Legend Snippet: Bacterial effector InlP binds host adaptor protein afadin. (A-B) InlP-GST fusion protein (InlP) or GST protein alone (-) bound to glutathione-Sepharose resin were incubated overnight with protein extracts from (A) MDCK or MDCK AF-6 -/- cells or (B) human placenta. Input (IN) and elution fractions (EF) were analyzed by Western blot with anti-afadin antibodies. Actin: loading control. (C-E) Immunofluorescence of human placental villi stained for afadin (red) and DAPI (blue). In panel C the white bar = 10 μm, the asterisk labels a portion of the stroma, and the arrow points to some of the CTBs. White rectangles in panel C indicate the locations of zoomed insets shown in panels D-E.

Techniques Used: Incubation, Western Blot, Immunofluorescence, Staining

20) Product Images from "Tpk3 and Snf1 protein kinases regulate Rgt1 association with Saccharomyces cerevisiae HXK2 promoter"

Article Title: Tpk3 and Snf1 protein kinases regulate Rgt1 association with Saccharomyces cerevisiae HXK2 promoter

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl028

Med8 and Rgt1 proteins interact at the HXK2 promoter level. ( A ) Model showing the three DNA fragments that were analysed: the downstream HXK2 regulatory region containing the Med8-binding site (266 bp), the upstream HXK2 regulatory region which contains the Rgt1 binding site (286 bp), and as a control, a region of 854 bp which contains both Med8 and Rgt1-binding sites. ( B ) Wild-type cells expressing HA-tagged Rgt1 or HA-tagged Med8 proteins (a) and untagged Rgt1 or Med8 proteins (b) were grown in low-glucose medium until an OD 600 of 1.0, lysed and subjected to ChIP. Genomic DNA was used as positive PCR control (lanes 1–3). Crosslinked-sonicated DNA (lanes 4–6) and immunoprecipitated DNA (lanes 7–11) were amplified by PCR using primer pairs spanning the RGT1 (7+8) and MED8 (9+10) elements of the HXK2 gene. These primer pairs had been defined previously to characterize Rgt1 and Med8 DNA binding. Wild-type cells expressing untagged Rgt1 or Med8 proteins were used in the ChIP experiment as negative controls (lanes 10 and 11). PCR products were resolved on a 2% agarose gel and visualized by ethidium bromide staining.
Figure Legend Snippet: Med8 and Rgt1 proteins interact at the HXK2 promoter level. ( A ) Model showing the three DNA fragments that were analysed: the downstream HXK2 regulatory region containing the Med8-binding site (266 bp), the upstream HXK2 regulatory region which contains the Rgt1 binding site (286 bp), and as a control, a region of 854 bp which contains both Med8 and Rgt1-binding sites. ( B ) Wild-type cells expressing HA-tagged Rgt1 or HA-tagged Med8 proteins (a) and untagged Rgt1 or Med8 proteins (b) were grown in low-glucose medium until an OD 600 of 1.0, lysed and subjected to ChIP. Genomic DNA was used as positive PCR control (lanes 1–3). Crosslinked-sonicated DNA (lanes 4–6) and immunoprecipitated DNA (lanes 7–11) were amplified by PCR using primer pairs spanning the RGT1 (7+8) and MED8 (9+10) elements of the HXK2 gene. These primer pairs had been defined previously to characterize Rgt1 and Med8 DNA binding. Wild-type cells expressing untagged Rgt1 or Med8 proteins were used in the ChIP experiment as negative controls (lanes 10 and 11). PCR products were resolved on a 2% agarose gel and visualized by ethidium bromide staining.

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Sonication, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining

Rgt1 DNA-binding activity is regulated by Tpk3 and Snf1 in vivo and in vitro . ( A ) Association of Rgt1 with the RGT1 element of the HXK2 promoter is detected by a ChIP assay. The W303-1A wild-type strain and the tpk12 , tpk13, tpk23 and snf1 mutant strains, transformed with the HA-Rgt1 construct, were grown in high-glucose (H-Glc) medium (b) until an OD 600 of 1.0 and then transferred to medium with low-glucose (L-Glc) for 60 min (a). The W303-1A wild-type strain was also transformed with an empty pWS93 plasmid to be used as a control with untagged Rgt1 (lane 6). The cells were treated with formaldehyde to cross-link proteins bound to DNA. ChIP was performed with anti-HA antibody. Input and immunoprecipitated DNA was amplified by PCR using primer pairs spanning the RGT1 element of the HXK2 gene promoter. PCR products were resolved on a 2% agarose gel and visualized by ethidium bromide staining. ( B ) EMSA was performed with probe RGT1 HXK2 by using nuclear extracts derived from exponentially growing wild-type cells, and snf1 , tpk1-2 and tpk2-3 mutant cells as indicated above the lanes. Lanes 1 and 10 are probe alone controls. CI, marks the position of the Rgt1-dependent shifted complex. F, free DNA.
Figure Legend Snippet: Rgt1 DNA-binding activity is regulated by Tpk3 and Snf1 in vivo and in vitro . ( A ) Association of Rgt1 with the RGT1 element of the HXK2 promoter is detected by a ChIP assay. The W303-1A wild-type strain and the tpk12 , tpk13, tpk23 and snf1 mutant strains, transformed with the HA-Rgt1 construct, were grown in high-glucose (H-Glc) medium (b) until an OD 600 of 1.0 and then transferred to medium with low-glucose (L-Glc) for 60 min (a). The W303-1A wild-type strain was also transformed with an empty pWS93 plasmid to be used as a control with untagged Rgt1 (lane 6). The cells were treated with formaldehyde to cross-link proteins bound to DNA. ChIP was performed with anti-HA antibody. Input and immunoprecipitated DNA was amplified by PCR using primer pairs spanning the RGT1 element of the HXK2 gene promoter. PCR products were resolved on a 2% agarose gel and visualized by ethidium bromide staining. ( B ) EMSA was performed with probe RGT1 HXK2 by using nuclear extracts derived from exponentially growing wild-type cells, and snf1 , tpk1-2 and tpk2-3 mutant cells as indicated above the lanes. Lanes 1 and 10 are probe alone controls. CI, marks the position of the Rgt1-dependent shifted complex. F, free DNA.

Techniques Used: Binding Assay, Activity Assay, In Vivo, In Vitro, Chromatin Immunoprecipitation, Mutagenesis, Transformation Assay, Construct, Gas Chromatography, Plasmid Preparation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Derivative Assay

Immunoprecipitation and GST pull-down assays of the interaction of Rgt1 with Hxk2 and Med8. ( A ) In vivo co-immunoprecipitation of Rgt1 with Hxk2. S.cerevisiae Y187 and the mutant strain hxk2Δ were transformed with HA-tagged Rgt1 protein. Cell extracts from the transformed Y187 wild-type strain grown in SD (high-glucose) medium, with selection for plasmid maintenance (lane 1), were immunoprecipitated with a polyclonal antibody to Pho4 (lane 2), or a polyclonal antibody to Hxk2 (lane 3). Cell extracts from the transformed hxk2Δ cells grown in SD (high-glucose) medium, with selection for plasmid maintenance, were immunoprecipitated with a polyclonal antibody to Hxk2 (lane 4). Immunoprecipitates were separated by 10% SDS–PAGE and co-precipitated HA-Rgt1 was visualized on a western blot with an anti-HA antibody. ( B ) GST pull-down assays of the interaction between Rgt1-Hxk2. Purified Rgt1 interacts with purified Hxk2. A GST-Hxk2 fusion protein was purified on glutathione–Sepharose columns and incubated with thrombin to isolate native Hxk2 (lane 1). Hxk2 was incubated with purified GST-Rgt1 or with GST on glutathione beads and washed extensively. Co-precipitated proteins were resolved on 10% SDS–PAGE. The Hxk2 protein was visualized on a western blot with anti-Hxk2 antibody. ( C ) Med8 protein co-precipitates together with Rgt1 from cell extracts. Extract from the yeast strain W303-1A (transformed with the plasmid pWS93/MED8), expressing the Med8-HA fusion protein, was incubated with GST-Rgt1 or with GST purified from E.coli on glutathione beads. The yeast cells were grown in SE (low-glucose) medium with selection for plasmid maintenance. Co-precipitated proteins were resolved on 12% SDS–PAGE. The Med8 protein was visualized on a western blot with an anti-HA antibody.
Figure Legend Snippet: Immunoprecipitation and GST pull-down assays of the interaction of Rgt1 with Hxk2 and Med8. ( A ) In vivo co-immunoprecipitation of Rgt1 with Hxk2. S.cerevisiae Y187 and the mutant strain hxk2Δ were transformed with HA-tagged Rgt1 protein. Cell extracts from the transformed Y187 wild-type strain grown in SD (high-glucose) medium, with selection for plasmid maintenance (lane 1), were immunoprecipitated with a polyclonal antibody to Pho4 (lane 2), or a polyclonal antibody to Hxk2 (lane 3). Cell extracts from the transformed hxk2Δ cells grown in SD (high-glucose) medium, with selection for plasmid maintenance, were immunoprecipitated with a polyclonal antibody to Hxk2 (lane 4). Immunoprecipitates were separated by 10% SDS–PAGE and co-precipitated HA-Rgt1 was visualized on a western blot with an anti-HA antibody. ( B ) GST pull-down assays of the interaction between Rgt1-Hxk2. Purified Rgt1 interacts with purified Hxk2. A GST-Hxk2 fusion protein was purified on glutathione–Sepharose columns and incubated with thrombin to isolate native Hxk2 (lane 1). Hxk2 was incubated with purified GST-Rgt1 or with GST on glutathione beads and washed extensively. Co-precipitated proteins were resolved on 10% SDS–PAGE. The Hxk2 protein was visualized on a western blot with anti-Hxk2 antibody. ( C ) Med8 protein co-precipitates together with Rgt1 from cell extracts. Extract from the yeast strain W303-1A (transformed with the plasmid pWS93/MED8), expressing the Med8-HA fusion protein, was incubated with GST-Rgt1 or with GST purified from E.coli on glutathione beads. The yeast cells were grown in SE (low-glucose) medium with selection for plasmid maintenance. Co-precipitated proteins were resolved on 12% SDS–PAGE. The Med8 protein was visualized on a western blot with an anti-HA antibody.

Techniques Used: Immunoprecipitation, In Vivo, Mutagenesis, Transformation Assay, Selection, Plasmid Preparation, SDS Page, Western Blot, Purification, Incubation, Expressing

21) Product Images from "PGC-1-Related Coactivator: Immediate Early Expression and Characterization of a CREB/NRF-1 Binding Domain Associated with Cytochrome c Promoter Occupancy and Respiratory Growth ▿"

Article Title: PGC-1-Related Coactivator: Immediate Early Expression and Characterization of a CREB/NRF-1 Binding Domain Associated with Cytochrome c Promoter Occupancy and Respiratory Growth ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00585-06

Deletion mapping the region of CREB required for binding PRC. A schematic representation of CREB is shown at top, with various functional domains indicated. Full-length CREB and C-terminal deletions, shown as solid lines below the diagram, were 35 S labeled and subjected to S-tag pulldown assays using either the upstream CREB binding domain in PRC (amino acids 400 to 604) (left panel) or the downstream CREB binding domain (amino acids 1379 to 1664) (right panel). Binding of the CREB subfragments to each PRC domain was compared to that of S-tagged thioredoxin as a negative control. Bound proteins were eluted from the washed S-protein agarose and visualized by autoradiography.
Figure Legend Snippet: Deletion mapping the region of CREB required for binding PRC. A schematic representation of CREB is shown at top, with various functional domains indicated. Full-length CREB and C-terminal deletions, shown as solid lines below the diagram, were 35 S labeled and subjected to S-tag pulldown assays using either the upstream CREB binding domain in PRC (amino acids 400 to 604) (left panel) or the downstream CREB binding domain (amino acids 1379 to 1664) (right panel). Binding of the CREB subfragments to each PRC domain was compared to that of S-tagged thioredoxin as a negative control. Bound proteins were eluted from the washed S-protein agarose and visualized by autoradiography.

Techniques Used: Binding Assay, Functional Assay, Labeling, Negative Control, Autoradiography

Comparison of the in vitro interactions between PRC and NRF-1, CREB, or HCF. A schematic representation of PRC is shown at top, with the various functional domains indicated (stippled, activation domain; cross-hatched, proline-rich region; gray, consensus recognition site [DHDY] for host cell factor [HCF]; black, R/S domain; vertical hatched, RNA recognition motif). Subfragments of PRC designated A, B, C, or D, with their amino acid coordinates shown in parentheses, were used in S-tag pulldown assays with 35 S-labeled transcription factors NRF-1, CREB, or HCF. Binding of the various subfragments to each 35 S-radiolabeled transcription factor was compared to that of S-tagged thioredoxin as a negative control. Bound proteins were eluted from the S-protein agarose and visualized by autoradiography.
Figure Legend Snippet: Comparison of the in vitro interactions between PRC and NRF-1, CREB, or HCF. A schematic representation of PRC is shown at top, with the various functional domains indicated (stippled, activation domain; cross-hatched, proline-rich region; gray, consensus recognition site [DHDY] for host cell factor [HCF]; black, R/S domain; vertical hatched, RNA recognition motif). Subfragments of PRC designated A, B, C, or D, with their amino acid coordinates shown in parentheses, were used in S-tag pulldown assays with 35 S-labeled transcription factors NRF-1, CREB, or HCF. Binding of the various subfragments to each 35 S-radiolabeled transcription factor was compared to that of S-tagged thioredoxin as a negative control. Bound proteins were eluted from the S-protein agarose and visualized by autoradiography.

Techniques Used: In Vitro, Functional Assay, Activation Assay, Labeling, Binding Assay, Negative Control, Autoradiography

In vivo interaction between PRC and CREB. (A) HA-tagged CREB was expressed in 293FT cells following electroporation with pSG5/CREB-HA. Cell extracts were immunoprecipitated with either 2.5 or 7.5 μg, respectively, of rabbit IgG as a negative control (lanes 1 and 2), anti-PRC(95-533) (lanes 3 and 4), or anti-PRC(1047-1379) (lanes 5 and 6). Immune complexes were brought down with protein A-agarose, washed, and run on an SDS-10% PAGE gel. For comparison, 20 μg of cell extract was run (lane 7). After transfer, the immunoblot was probed with mouse anti-HA monoclonal antibody. (B) 293FT cell extracts were immunoprecipitated with either 2.5 or 7.5 μg rabbit IgG as a negative control (lanes 1 and 2) or anti-PRC(1047-1379) (lanes 3 and 4). Immune complexes were precipitated and electroblotted as described for panel A. For comparison, 2 ng of recombinant NRF-1 was run (lane 5). After transfer, the immunoblot was probed with goat anti-NRF-1. Molecular mass standards in kilodaltons are indicated at the left in each panel.
Figure Legend Snippet: In vivo interaction between PRC and CREB. (A) HA-tagged CREB was expressed in 293FT cells following electroporation with pSG5/CREB-HA. Cell extracts were immunoprecipitated with either 2.5 or 7.5 μg, respectively, of rabbit IgG as a negative control (lanes 1 and 2), anti-PRC(95-533) (lanes 3 and 4), or anti-PRC(1047-1379) (lanes 5 and 6). Immune complexes were brought down with protein A-agarose, washed, and run on an SDS-10% PAGE gel. For comparison, 20 μg of cell extract was run (lane 7). After transfer, the immunoblot was probed with mouse anti-HA monoclonal antibody. (B) 293FT cell extracts were immunoprecipitated with either 2.5 or 7.5 μg rabbit IgG as a negative control (lanes 1 and 2) or anti-PRC(1047-1379) (lanes 3 and 4). Immune complexes were precipitated and electroblotted as described for panel A. For comparison, 2 ng of recombinant NRF-1 was run (lane 5). After transfer, the immunoblot was probed with goat anti-NRF-1. Molecular mass standards in kilodaltons are indicated at the left in each panel.

Techniques Used: In Vivo, Electroporation, Immunoprecipitation, Negative Control, Polyacrylamide Gel Electrophoresis, Recombinant

22) Product Images from "OsSpo11-4, a Rice Homologue of the Archaeal TopVIA Protein, Mediates Double-Strand DNA Cleavage and Interacts with OsTopVIB"

Article Title: OsSpo11-4, a Rice Homologue of the Archaeal TopVIA Protein, Mediates Double-Strand DNA Cleavage and Interacts with OsTopVIB

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020327

Double-strand DNA cleavage catalyzed by purified OsSpo11 and OsTopVIB proteins. Each purified OsSpo11 and OsTopVIB protein or a combination shown above the image was added into a reaction mixture containing substrate, and purified GST was used as a control. After reaction, the mixture was subjected to agarose separation (for details, see “ Materials and methods ”). dkDNA, decatenated kDNA Marker; lkDNA, linear kDNA marker; kDNA, kinetoplast DNA; s5, OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, TopVIB; GST, the GST protein control. A , kDNA as a substrate. dkDNA shows relative positions of open circular nicked DNA—OC, and relaxed, closed circular monomers—CC; dkDNA and lkDNA markers were provided by the Topoisomerase Assay Kit, kDNA was used as a catenated DNA reference after incubation in a reaction mixture without protein. B , pUC18 plasmid as a substrate. pUC18 refers to a reaction containing buffer and pUC18 plasmids only (c, circular pUC18 marker). Eco RI refers to pUC18 plasmids digested by Eco RI, which cuts pUC18 only once (l, linear pUC18 marker). C , DNA cleavage reaction rate is proportioned to the concentration of OsSpo11-4. s4 refers to only reaction buffer and OsSpo11-4 were added, while 1∼6 refer to 0.5 µM, 0.4 µM, 0.3 µM, 0.2 µM, 0.1 µM and 0 µM OsSpo11-4 were added to the standard reaction, respectively. Cleavage activity was determined using kDNA decatenation assays. D , the effect of Mg 2+ on OsSpo11-4 activity. 0∼5 refer to 0 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM and 12.5 mM Mg 2 were added to the standard reaction (kDNA decatenation), respectively. E , reaction rate quantification of 0∼5 in panel D. Reaction rate was determined as a percentage of linear kDNA generated compared to the total kDNA added.
Figure Legend Snippet: Double-strand DNA cleavage catalyzed by purified OsSpo11 and OsTopVIB proteins. Each purified OsSpo11 and OsTopVIB protein or a combination shown above the image was added into a reaction mixture containing substrate, and purified GST was used as a control. After reaction, the mixture was subjected to agarose separation (for details, see “ Materials and methods ”). dkDNA, decatenated kDNA Marker; lkDNA, linear kDNA marker; kDNA, kinetoplast DNA; s5, OsSpo11-5; s1, OsSpo11-1; s4, OsSpo11-4; VIB, TopVIB; GST, the GST protein control. A , kDNA as a substrate. dkDNA shows relative positions of open circular nicked DNA—OC, and relaxed, closed circular monomers—CC; dkDNA and lkDNA markers were provided by the Topoisomerase Assay Kit, kDNA was used as a catenated DNA reference after incubation in a reaction mixture without protein. B , pUC18 plasmid as a substrate. pUC18 refers to a reaction containing buffer and pUC18 plasmids only (c, circular pUC18 marker). Eco RI refers to pUC18 plasmids digested by Eco RI, which cuts pUC18 only once (l, linear pUC18 marker). C , DNA cleavage reaction rate is proportioned to the concentration of OsSpo11-4. s4 refers to only reaction buffer and OsSpo11-4 were added, while 1∼6 refer to 0.5 µM, 0.4 µM, 0.3 µM, 0.2 µM, 0.1 µM and 0 µM OsSpo11-4 were added to the standard reaction, respectively. Cleavage activity was determined using kDNA decatenation assays. D , the effect of Mg 2+ on OsSpo11-4 activity. 0∼5 refer to 0 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM and 12.5 mM Mg 2 were added to the standard reaction (kDNA decatenation), respectively. E , reaction rate quantification of 0∼5 in panel D. Reaction rate was determined as a percentage of linear kDNA generated compared to the total kDNA added.

Techniques Used: Purification, Marker, Incubation, Plasmid Preparation, Concentration Assay, Activity Assay, Generated

Expression and purification of OsSpo11 and OsTopVIB proteins. Proteins were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue (A∼C) or hybridized with an antibody against GST (D). A , Expression of the 4 proteins each in S. prombe cells (OsSpo11-4 showed here as an example). Cells harboring recombinant plasmids were treated with (+vb1) or without (−vb1) vitamin B1. GST-OsSpo11-4 fusion protein from −vb1 was purified with Glutathione Sepharose 4B resin. B , Removal of GST tag from purified GST-protein fusion by thrombin digestion (OsSpo11-4 shown as an example). The reaction mixture was incubated for different times shown above the image and subjected to SDS-PAGE separation. C , Purified GST-tagged OsSpo11s and OsTopVIB. D , Western blot analysis of purified GST-tagged proteins in C with a monoclonal antibody against GST. MW, molecular weight standards.
Figure Legend Snippet: Expression and purification of OsSpo11 and OsTopVIB proteins. Proteins were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue (A∼C) or hybridized with an antibody against GST (D). A , Expression of the 4 proteins each in S. prombe cells (OsSpo11-4 showed here as an example). Cells harboring recombinant plasmids were treated with (+vb1) or without (−vb1) vitamin B1. GST-OsSpo11-4 fusion protein from −vb1 was purified with Glutathione Sepharose 4B resin. B , Removal of GST tag from purified GST-protein fusion by thrombin digestion (OsSpo11-4 shown as an example). The reaction mixture was incubated for different times shown above the image and subjected to SDS-PAGE separation. C , Purified GST-tagged OsSpo11s and OsTopVIB. D , Western blot analysis of purified GST-tagged proteins in C with a monoclonal antibody against GST. MW, molecular weight standards.

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

Seed setting (A, B), pollen viability (C) and endogenous OsSpo11-4 expression (D) were decreased in OsSpo11-4 RNAi lines. A , Panicle morphology of wild-type (left) and OsSpo11-4 RNAi T0 lines with different seed setting (right 3). B , Seed setting of wild-type (WT) and 6 RNAi T1 generation lines (L11, L19, L8, L38, L39 and L45). C , Alexander staining of anthers (a and b) and mature pollen grains (c and d) from wild-type (a and c) and RNAi (b and d) plants showing reduced pollen viability in RNAi lines. Scale bar = 100 µm in (a) and (b), and 50 µm in (c) and (d). D , Semi-quantitative RT-PCR analysis of endogenous OsSpo11-4 mRNA levels in wild-type and RNAi lines. TubA mRNA was amplified as an internal control. All PCR products were separated on 1% agarose gel. E , Quantitative real-time RT-PCR analysis for expression of OsSpo11-4 in wild type and RNAi lines. The expression levels of SPO11-4 in different RNAi lines were firstly normalized by computing to the internal standard gene, tubA , and then compared to the wild type by the ΔΔC T method.
Figure Legend Snippet: Seed setting (A, B), pollen viability (C) and endogenous OsSpo11-4 expression (D) were decreased in OsSpo11-4 RNAi lines. A , Panicle morphology of wild-type (left) and OsSpo11-4 RNAi T0 lines with different seed setting (right 3). B , Seed setting of wild-type (WT) and 6 RNAi T1 generation lines (L11, L19, L8, L38, L39 and L45). C , Alexander staining of anthers (a and b) and mature pollen grains (c and d) from wild-type (a and c) and RNAi (b and d) plants showing reduced pollen viability in RNAi lines. Scale bar = 100 µm in (a) and (b), and 50 µm in (c) and (d). D , Semi-quantitative RT-PCR analysis of endogenous OsSpo11-4 mRNA levels in wild-type and RNAi lines. TubA mRNA was amplified as an internal control. All PCR products were separated on 1% agarose gel. E , Quantitative real-time RT-PCR analysis for expression of OsSpo11-4 in wild type and RNAi lines. The expression levels of SPO11-4 in different RNAi lines were firstly normalized by computing to the internal standard gene, tubA , and then compared to the wild type by the ΔΔC T method.

Techniques Used: Expressing, Staining, Quantitative RT-PCR, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

23) Product Images from "ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †"

Article Title: ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.12.5235-5248.2004

Mapping regions of ABIN-2 which interact with p105 and TPL-2. (A) Schematic diagram of recombinant GST-ABIN-2 fusion proteins. The positions of the ABIN homology domain (AHD) and the binding regions for TPL-2, p105, and A20 are shown. The N and C termini of the wild-type (WT) GST-ABIN-2 protein (amino acids 1 to 420) are indicated. (B and C) 293 cells were transfected with vectors encoding Myc-p105, Myc-TPL-2, or Myc-A20. Cell lysates were prepared using 1% Brij 58 buffer A and incubated with the indicated GST-ABIN-2 fusion proteins or GST (control) coupled to glutathione-Sepharose 4B. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.
Figure Legend Snippet: Mapping regions of ABIN-2 which interact with p105 and TPL-2. (A) Schematic diagram of recombinant GST-ABIN-2 fusion proteins. The positions of the ABIN homology domain (AHD) and the binding regions for TPL-2, p105, and A20 are shown. The N and C termini of the wild-type (WT) GST-ABIN-2 protein (amino acids 1 to 420) are indicated. (B and C) 293 cells were transfected with vectors encoding Myc-p105, Myc-TPL-2, or Myc-A20. Cell lysates were prepared using 1% Brij 58 buffer A and incubated with the indicated GST-ABIN-2 fusion proteins or GST (control) coupled to glutathione-Sepharose 4B. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.

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

Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2 1-429 fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105 497-968 fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [ 35 S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2 1-429 was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2 398-467 peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.
Figure Legend Snippet: Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2 1-429 fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105 497-968 fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [ 35 S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2 1-429 was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2 398-467 peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.

Techniques Used: Transfection, Mutagenesis, Incubation, Affinity Purification, SDS Page, Western Blot, Labeling, In Vitro, Isolation, Autoradiography

ABIN-2 preferentially interacts with a p105/TPL-2 complex. (A and B) Duplicate cultures of 293 cells were cotransfected with vectors encoding ABIN2-FL and HA-p105 or Myc-TPL-2 or with EV. Cell lysates were prepared from each duplicate culture set using either buffer A (1% NP-40) or RIPA buffer, as indicated. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). HA-p105 and Myc-TPL-2 mRNA levels in total RNA were assayed by semiquantitative RT-PCR (bottom blots). The 18S rRNA amplicon was used as an internal control. (C) 293 cells were cotransfected with vectors encoding HA-p105 and TPL-2 individually or together. Transfected proteins were affinity purified from cell lysates, prepared in 1% NP-40 buffer A, using GST-ABIN-2 1-429 fusion protein coupled to glutathione-Sepharose. Isolated proteins were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.
Figure Legend Snippet: ABIN-2 preferentially interacts with a p105/TPL-2 complex. (A and B) Duplicate cultures of 293 cells were cotransfected with vectors encoding ABIN2-FL and HA-p105 or Myc-TPL-2 or with EV. Cell lysates were prepared from each duplicate culture set using either buffer A (1% NP-40) or RIPA buffer, as indicated. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). HA-p105 and Myc-TPL-2 mRNA levels in total RNA were assayed by semiquantitative RT-PCR (bottom blots). The 18S rRNA amplicon was used as an internal control. (C) 293 cells were cotransfected with vectors encoding HA-p105 and TPL-2 individually or together. Transfected proteins were affinity purified from cell lysates, prepared in 1% NP-40 buffer A, using GST-ABIN-2 1-429 fusion protein coupled to glutathione-Sepharose. Isolated proteins were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.

Techniques Used: SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, Affinity Purification, Isolation

24) Product Images from "ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †"

Article Title: ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.12.5235-5248.2004

Mapping regions of ABIN-2 which interact with p105 and TPL-2. (A) Schematic diagram of recombinant GST-ABIN-2 fusion proteins. The positions of the ABIN homology domain (AHD) and the binding regions for TPL-2, p105, and A20 are shown. The N and C termini of the wild-type (WT) GST-ABIN-2 protein (amino acids 1 to 420) are indicated. (B and C) 293 cells were transfected with vectors encoding Myc-p105, Myc-TPL-2, or Myc-A20. Cell lysates were prepared using 1% Brij 58 buffer A and incubated with the indicated GST-ABIN-2 fusion proteins or GST (control) coupled to glutathione-Sepharose 4B. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.
Figure Legend Snippet: Mapping regions of ABIN-2 which interact with p105 and TPL-2. (A) Schematic diagram of recombinant GST-ABIN-2 fusion proteins. The positions of the ABIN homology domain (AHD) and the binding regions for TPL-2, p105, and A20 are shown. The N and C termini of the wild-type (WT) GST-ABIN-2 protein (amino acids 1 to 420) are indicated. (B and C) 293 cells were transfected with vectors encoding Myc-p105, Myc-TPL-2, or Myc-A20. Cell lysates were prepared using 1% Brij 58 buffer A and incubated with the indicated GST-ABIN-2 fusion proteins or GST (control) coupled to glutathione-Sepharose 4B. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.

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

Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2 1-429 fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105 497-968 fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [ 35 S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2 1-429 was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2 398-467 peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.
Figure Legend Snippet: Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2 1-429 fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105 497-968 fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [ 35 S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2 1-429 was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2 398-467 peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.

Techniques Used: Transfection, Mutagenesis, Incubation, Affinity Purification, SDS Page, Western Blot, Labeling, In Vitro, Isolation, Autoradiography

ABIN-2 preferentially interacts with a p105/TPL-2 complex. (A and B) Duplicate cultures of 293 cells were cotransfected with vectors encoding ABIN2-FL and HA-p105 or Myc-TPL-2 or with EV. Cell lysates were prepared from each duplicate culture set using either buffer A (1% NP-40) or RIPA buffer, as indicated. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). HA-p105 and Myc-TPL-2 mRNA levels in total RNA were assayed by semiquantitative RT-PCR (bottom blots). The 18S rRNA amplicon was used as an internal control. (C) 293 cells were cotransfected with vectors encoding HA-p105 and TPL-2 individually or together. Transfected proteins were affinity purified from cell lysates, prepared in 1% NP-40 buffer A, using GST-ABIN-2 1-429 fusion protein coupled to glutathione-Sepharose. Isolated proteins were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.
Figure Legend Snippet: ABIN-2 preferentially interacts with a p105/TPL-2 complex. (A and B) Duplicate cultures of 293 cells were cotransfected with vectors encoding ABIN2-FL and HA-p105 or Myc-TPL-2 or with EV. Cell lysates were prepared from each duplicate culture set using either buffer A (1% NP-40) or RIPA buffer, as indicated. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). HA-p105 and Myc-TPL-2 mRNA levels in total RNA were assayed by semiquantitative RT-PCR (bottom blots). The 18S rRNA amplicon was used as an internal control. (C) 293 cells were cotransfected with vectors encoding HA-p105 and TPL-2 individually or together. Transfected proteins were affinity purified from cell lysates, prepared in 1% NP-40 buffer A, using GST-ABIN-2 1-429 fusion protein coupled to glutathione-Sepharose. Isolated proteins were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.

Techniques Used: SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, Affinity Purification, Isolation

25) Product Images from "Both Ser361 phosphorylation and the C‐arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint"

Article Title: Both Ser361 phosphorylation and the C‐arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12518

Analysis of TXNIP phosphorylation sites. (A) Structure of TXNIP. Two α‐arrestin domains and putative phosphorylation sites analyzed in this study are indicated. Phosphopeptides analyzed by LC‐MS/MS are indicated with gray boxes. (B) LC‐MS/MS analysis of TXNIP overexpressed in COS‐7 cells. Affinity‐purified FLAG–TXNIP protein was in‐gel digested by trypsin, chymotrypsin, and aspartic protease, and its phosphorylation state was analyzed by mass spectrometry. Phosphorylation of Ser314, Ser346, Thr349, and Ser361 was detected. (C) COS‐7 cells were transfected with each expression plasmid as indicated, and cell lysate was immunoprecipitated with anti‐FLAG agarose gel. The phosphorylation state of TXNIP and its mutants were analyzed using Pro‐Q phosphoprotein gel stain. The intensity of phosphorylation bands was shown as mean ± SD ( t test, n = 4).
Figure Legend Snippet: Analysis of TXNIP phosphorylation sites. (A) Structure of TXNIP. Two α‐arrestin domains and putative phosphorylation sites analyzed in this study are indicated. Phosphopeptides analyzed by LC‐MS/MS are indicated with gray boxes. (B) LC‐MS/MS analysis of TXNIP overexpressed in COS‐7 cells. Affinity‐purified FLAG–TXNIP protein was in‐gel digested by trypsin, chymotrypsin, and aspartic protease, and its phosphorylation state was analyzed by mass spectrometry. Phosphorylation of Ser314, Ser346, Thr349, and Ser361 was detected. (C) COS‐7 cells were transfected with each expression plasmid as indicated, and cell lysate was immunoprecipitated with anti‐FLAG agarose gel. The phosphorylation state of TXNIP and its mutants were analyzed using Pro‐Q phosphoprotein gel stain. The intensity of phosphorylation bands was shown as mean ± SD ( t test, n = 4).

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Affinity Purification, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Agarose Gel Electrophoresis, Staining

TXNIP phosphorylation at Ser361 promotes the interaction with JAB1. (A, B) COS‐7 cells were cotransfected by expression plasmids for FLAG–TXNIP (wild‐type (WT), FLAG–T349A or FLAG–S361A) and JAB1–V5 (A), myc‐p27 kip1 , Cdk2–V5, or cyclin E–V5 (B), as indicated. Protein complex was immunoprecipitated by anti‐V5, anti‐myc or anti‐FLAG agarose, and the protein interaction was analyzed by western blot analysis. Error bars in the graphs indicate mean ± SD. (C) COS‐7 cells were cotransfected by expression plasmids for myc‐p27 kip1 and FLAG–TXNIP (wild‐type, FLAG–T349A or FLAG–S361A). Protein complex was immunoprecipitated by different types of antibodies as indicated in the figure. Western blot analysis was used to analyze the interaction of p27 kip1 and JAB1. Signal densities of JAB1 immunoprecipitated with p27 kip1 were analyzed. For each value, difference from the control (without TXNIP transfection) was calculated and expressed as mean ± SD (* P
Figure Legend Snippet: TXNIP phosphorylation at Ser361 promotes the interaction with JAB1. (A, B) COS‐7 cells were cotransfected by expression plasmids for FLAG–TXNIP (wild‐type (WT), FLAG–T349A or FLAG–S361A) and JAB1–V5 (A), myc‐p27 kip1 , Cdk2–V5, or cyclin E–V5 (B), as indicated. Protein complex was immunoprecipitated by anti‐V5, anti‐myc or anti‐FLAG agarose, and the protein interaction was analyzed by western blot analysis. Error bars in the graphs indicate mean ± SD. (C) COS‐7 cells were cotransfected by expression plasmids for myc‐p27 kip1 and FLAG–TXNIP (wild‐type, FLAG–T349A or FLAG–S361A). Protein complex was immunoprecipitated by different types of antibodies as indicated in the figure. Western blot analysis was used to analyze the interaction of p27 kip1 and JAB1. Signal densities of JAB1 immunoprecipitated with p27 kip1 were analyzed. For each value, difference from the control (without TXNIP transfection) was calculated and expressed as mean ± SD (* P

Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection

TXNIP is phosphorylated by p38 MAPK. (A) Phosphorylation of TXNIP in COS‐7 cells and HuH‐7 cells. COS‐7 cells were transfected with expression plasmid for FLAG–TXNIP, and cell lysate was immunoprecipitated with anti‐FLAG agarose gel. HuH‐7 cells were treated with 50 m m d ‐allose for 48 h, and cell lysate was immunoprecipitated with anti‐TXNIP/protein G, and separated by SDS/PAGE. The phosphorylation of TXNIP was detected by Pro‐Q phosphoprotein gel stain. (B) In vitro phosphorylation analysis of TXNIP by autoradiography. Affinity‐purified FLAG–TXNIP protein was incubated in the presence of each kinase and γ‐[ 32 P]ATP, and phosphorylated TXNIP was detected. (C) Phosphorylation of TXNIP by p38 MAPK in COS‐7 cells. COS‐7 cells were transfected with expression plasmid for FLAG–TXNIP and p38 MAPK–V5. Cells were pretreated with LY2228820 (500 n m ; 2 h) as indicated. The phosphorylation of TXNIP was analyzed by immunoprecipitation followed by the Pro‐Q phosphoprotein gel stain. (D) Phosphorylation of TXNIP by kinases in COS‐7 cells. COS‐7 cells were cotransfected with expression plasmids for FLAG–TXNIP and each V5‐tagged kinase. The phosphorylation of TXNIP was analyzed by immunoprecipitation followed by Pro‐Q phosphoprotein gel stain.
Figure Legend Snippet: TXNIP is phosphorylated by p38 MAPK. (A) Phosphorylation of TXNIP in COS‐7 cells and HuH‐7 cells. COS‐7 cells were transfected with expression plasmid for FLAG–TXNIP, and cell lysate was immunoprecipitated with anti‐FLAG agarose gel. HuH‐7 cells were treated with 50 m m d ‐allose for 48 h, and cell lysate was immunoprecipitated with anti‐TXNIP/protein G, and separated by SDS/PAGE. The phosphorylation of TXNIP was detected by Pro‐Q phosphoprotein gel stain. (B) In vitro phosphorylation analysis of TXNIP by autoradiography. Affinity‐purified FLAG–TXNIP protein was incubated in the presence of each kinase and γ‐[ 32 P]ATP, and phosphorylated TXNIP was detected. (C) Phosphorylation of TXNIP by p38 MAPK in COS‐7 cells. COS‐7 cells were transfected with expression plasmid for FLAG–TXNIP and p38 MAPK–V5. Cells were pretreated with LY2228820 (500 n m ; 2 h) as indicated. The phosphorylation of TXNIP was analyzed by immunoprecipitation followed by the Pro‐Q phosphoprotein gel stain. (D) Phosphorylation of TXNIP by kinases in COS‐7 cells. COS‐7 cells were cotransfected with expression plasmids for FLAG–TXNIP and each V5‐tagged kinase. The phosphorylation of TXNIP was analyzed by immunoprecipitation followed by Pro‐Q phosphoprotein gel stain.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Agarose Gel Electrophoresis, SDS Page, Staining, In Vitro, Autoradiography, Affinity Purification, Incubation

26) Product Images from "XIAP mediates NOD signaling via interaction with RIP2"

Article Title: XIAP mediates NOD signaling via interaction with RIP2

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

doi: 10.1073/pnas.0907131106

XIAP protein associates with the NOD/RIP2 complex. Myc-NOD1 ( A ) or Myc-NOD2 ( B ) were expressed in HEK293T cells along with GFP-RIP2 (wild-type [WT]), GFP-RIP2ΔCARD or GFP-RIP2Δkinase domain (KD). Protein lysates (1 mg) were incubated with GST-XIAP immobilized on glutathione-Sepharose and adsorbed proteins were analyzed by immunoblotting using anti-Myc and anti-GFP antibodies. An aliquot of lysates (input) was analyzed directly by immunoblotting.
Figure Legend Snippet: XIAP protein associates with the NOD/RIP2 complex. Myc-NOD1 ( A ) or Myc-NOD2 ( B ) were expressed in HEK293T cells along with GFP-RIP2 (wild-type [WT]), GFP-RIP2ΔCARD or GFP-RIP2Δkinase domain (KD). Protein lysates (1 mg) were incubated with GST-XIAP immobilized on glutathione-Sepharose and adsorbed proteins were analyzed by immunoblotting using anti-Myc and anti-GFP antibodies. An aliquot of lysates (input) was analyzed directly by immunoblotting.

Techniques Used: Incubation

SMAC binding site of BIR2 domain of XIAP is required for RIP2 binding. ( A ) Schematic representation of GFP-XIAP mutants. ( B ) Transfected HEK293T cells expressing FLAG-RIP2 together with GFP-XIAP WT , GFP-XIAP E219R , GFP-XIAP H223V , GFP-XIAP E219R/H223V or GFP-control were lysed and subjected to immunoprecipitation using anti-FLAG antibody. Immunoprecipitates were analyzed by SDS/PAGE/immunoblotting using anti-FLAG and anti-GFP antibodies. Protein binding was quantified by densitometry analysis, measuring the integrated density value expressed as arbitrary units of the GFP-XIAP bands. Values are expressed as mean ± SD of three independent experiments. ( C–E ) Lysates (1 mg) of transfected HEK293T cells expressing FLAG-RIP2 were incubated with 2 μg of recombinant GST-XIAP immobilized on glutathione-Sepharose along with various amounts of His-6-SMAC protein C , SMAC peptide ( D ), or SMAC-mimicking compounds ABT-10, nonSMAC-mimicking compound TPI-1396–11, or vehicle control ( E ). Beads were analyzed by immunoblotting using anti-FLAG-HRP, anti-XIAP/anti-GST or anti-SMAC antibodies as indicated. An aliquot of lysates was also directly analyzed by immunoblotting (“input”).
Figure Legend Snippet: SMAC binding site of BIR2 domain of XIAP is required for RIP2 binding. ( A ) Schematic representation of GFP-XIAP mutants. ( B ) Transfected HEK293T cells expressing FLAG-RIP2 together with GFP-XIAP WT , GFP-XIAP E219R , GFP-XIAP H223V , GFP-XIAP E219R/H223V or GFP-control were lysed and subjected to immunoprecipitation using anti-FLAG antibody. Immunoprecipitates were analyzed by SDS/PAGE/immunoblotting using anti-FLAG and anti-GFP antibodies. Protein binding was quantified by densitometry analysis, measuring the integrated density value expressed as arbitrary units of the GFP-XIAP bands. Values are expressed as mean ± SD of three independent experiments. ( C–E ) Lysates (1 mg) of transfected HEK293T cells expressing FLAG-RIP2 were incubated with 2 μg of recombinant GST-XIAP immobilized on glutathione-Sepharose along with various amounts of His-6-SMAC protein C , SMAC peptide ( D ), or SMAC-mimicking compounds ABT-10, nonSMAC-mimicking compound TPI-1396–11, or vehicle control ( E ). Beads were analyzed by immunoblotting using anti-FLAG-HRP, anti-XIAP/anti-GST or anti-SMAC antibodies as indicated. An aliquot of lysates was also directly analyzed by immunoblotting (“input”).

Techniques Used: Binding Assay, Transfection, Expressing, Immunoprecipitation, SDS Page, Protein Binding, Incubation, Recombinant

XIAP binds RIP2. ( A ) HEK293T cells were co-transfected with plasmids encoding FLAG-XIAP, GFP-RIP2 WT , GFP-RIP2 ΔCARD , GFP-RIP2 Δkinase domain (KD) or empty pEGFP-C2, as indicated. After 24 h, cell lysates were prepared, normalized for protein content, and GFP-tagged proteins were immunoprecipitated using anti-GFP antibody. Immunoprecipitates were analyzed by immunoblotting using antibodies specific for FLAG epitope (Top) or GFP ( Middle ). Alternatively, cell lysates were analyzed directly by SDS/PAGE/immunoblotting ( Bottom ). Molecular weight (MW) markers are indicated in kilo-Daltons (kDa). (*HC and *LC indicate Ig heavy and light chains). ( B ) Lysates of THP-1 cells were immunoprecipitated with control IgG or rat anti-RIP2 antibody. The resulting immunoprecipitates were analyzed by immunoblotting using mouse monoclonal anti-XIAP antibody (Top) . The cell lysate (50 μg protein) was also analyzed by SDS/PAGE/immunoblotting using mouse-monoclonal anti-XIAP or rat monoclonal anti-RIP2 ( Bottom ). ( C ) Lysates of transfected HEK293T cells expressing FLAG-RIP2 were incubated with recombinant GST-XIAP, various GST-XIAP fragments, or GST-Survivin immobilized on glutathione Sepharose and bound proteins were analyzed by SDS/PAGE/immunoblotting using mouse monoclonal anti-FLAG (Top) and anti-GST ( Bottom ) antibodies. Asterisks denote nonspecific bands.
Figure Legend Snippet: XIAP binds RIP2. ( A ) HEK293T cells were co-transfected with plasmids encoding FLAG-XIAP, GFP-RIP2 WT , GFP-RIP2 ΔCARD , GFP-RIP2 Δkinase domain (KD) or empty pEGFP-C2, as indicated. After 24 h, cell lysates were prepared, normalized for protein content, and GFP-tagged proteins were immunoprecipitated using anti-GFP antibody. Immunoprecipitates were analyzed by immunoblotting using antibodies specific for FLAG epitope (Top) or GFP ( Middle ). Alternatively, cell lysates were analyzed directly by SDS/PAGE/immunoblotting ( Bottom ). Molecular weight (MW) markers are indicated in kilo-Daltons (kDa). (*HC and *LC indicate Ig heavy and light chains). ( B ) Lysates of THP-1 cells were immunoprecipitated with control IgG or rat anti-RIP2 antibody. The resulting immunoprecipitates were analyzed by immunoblotting using mouse monoclonal anti-XIAP antibody (Top) . The cell lysate (50 μg protein) was also analyzed by SDS/PAGE/immunoblotting using mouse-monoclonal anti-XIAP or rat monoclonal anti-RIP2 ( Bottom ). ( C ) Lysates of transfected HEK293T cells expressing FLAG-RIP2 were incubated with recombinant GST-XIAP, various GST-XIAP fragments, or GST-Survivin immobilized on glutathione Sepharose and bound proteins were analyzed by SDS/PAGE/immunoblotting using mouse monoclonal anti-FLAG (Top) and anti-GST ( Bottom ) antibodies. Asterisks denote nonspecific bands.

Techniques Used: Transfection, Immunoprecipitation, FLAG-tag, SDS Page, Molecular Weight, Expressing, Incubation, Recombinant

27) Product Images from "Purification of Capping Protein Using the Capping Protein Binding Site of CARMIL as an Affinity Matrix"

Article Title: Purification of Capping Protein Using the Capping Protein Binding Site of CARMIL as an Affinity Matrix

Journal: Protein expression and purification

doi: 10.1016/j.pep.2009.05.002

Quantitative immunoblot analyses of the distributions of CP in ammonium sulfate fractions and small scale CP purifications using Acanthamoeba HSS The HSS of lysed Acanthamoeba was subjected to ammonium sulfate fractionation (Panel A: 0–25% pellet, 25–55% pellet and 55% supernatant in lanes 2, 4 and 3, respectively), a single ammonium sulfate precipitation (Panel B: 0–55% supernatant and 0–55% pellet in lanes 2 and 3, respectively), or not fractionated (Panel C). The 0–55% supernatant in Panel B was then subjected to small scale affinity chromatography on GST-AP Sepharose, and the unbound and bound fractions obtained (Panel B, lanes 4 and 5, respectively). Similarly, the HSS in Panel C was subjected to small scale affinity chromatography on GST-AP Sepharose, and the unbound and bound fractions obtained (Panel C, lanes 2 and 3, respectively). All of the samples in Panels AC were then probed by Western blotting using a polyclonal antibody raised against the α-subunit of Acanthamoeba ]. To allow the estimation of the relative distribution of CP in various ammonium sulfate fractions and in bound versus unbound fractions, the percent of the total fraction loaded was the same for the samples in lanes 2–4 in Panel A, for the samples in lanes 2–3 in Panel B, for the samples in lanes 4 and 5 in Panel B, and for the samples in lanes 2 and 3 in Panel C.
Figure Legend Snippet: Quantitative immunoblot analyses of the distributions of CP in ammonium sulfate fractions and small scale CP purifications using Acanthamoeba HSS The HSS of lysed Acanthamoeba was subjected to ammonium sulfate fractionation (Panel A: 0–25% pellet, 25–55% pellet and 55% supernatant in lanes 2, 4 and 3, respectively), a single ammonium sulfate precipitation (Panel B: 0–55% supernatant and 0–55% pellet in lanes 2 and 3, respectively), or not fractionated (Panel C). The 0–55% supernatant in Panel B was then subjected to small scale affinity chromatography on GST-AP Sepharose, and the unbound and bound fractions obtained (Panel B, lanes 4 and 5, respectively). Similarly, the HSS in Panel C was subjected to small scale affinity chromatography on GST-AP Sepharose, and the unbound and bound fractions obtained (Panel C, lanes 2 and 3, respectively). All of the samples in Panels AC were then probed by Western blotting using a polyclonal antibody raised against the α-subunit of Acanthamoeba ]. To allow the estimation of the relative distribution of CP in various ammonium sulfate fractions and in bound versus unbound fractions, the percent of the total fraction loaded was the same for the samples in lanes 2–4 in Panel A, for the samples in lanes 2–3 in Panel B, for the samples in lanes 4 and 5 in Panel B, and for the samples in lanes 2 and 3 in Panel C.

Techniques Used: Fractionation, Affinity Chromatography, Western Blot

Generation of the GST-AP affinity matrix Fractions sampled during the procedure were resolved by SDS-PAGE and stained with Coomassie Blue. Shown are lysates of E. coli harboring the GST-AP plasmid before (lane 1) and 3 h after induction of protein expression (lane 2), as well as the HSS (i.e. the soluble fraction) (lane 3) that was then captured on Glutathione Sepharose beads. After elution of GST-AP (lane 4) and dialysis against coupling buffer, the fusion protein was immobilized on CNBr-activated Sepharose. Coupling appeared to be complete, as the flow through from the coupling reaction (lane 5) was devoid of GST-AP. M, molecular mass standards.
Figure Legend Snippet: Generation of the GST-AP affinity matrix Fractions sampled during the procedure were resolved by SDS-PAGE and stained with Coomassie Blue. Shown are lysates of E. coli harboring the GST-AP plasmid before (lane 1) and 3 h after induction of protein expression (lane 2), as well as the HSS (i.e. the soluble fraction) (lane 3) that was then captured on Glutathione Sepharose beads. After elution of GST-AP (lane 4) and dialysis against coupling buffer, the fusion protein was immobilized on CNBr-activated Sepharose. Coupling appeared to be complete, as the flow through from the coupling reaction (lane 5) was devoid of GST-AP. M, molecular mass standards.

Techniques Used: SDS Page, Staining, Plasmid Preparation, Expressing, Flow Cytometry

Large scale purification of mouse CP from bacterial lysates using GST-mCAH3 as an affinity matrix Samples taken during the purification procedure were separated by SDS-PAGE and stained with Coomassie Blue. Shown are the HSS of lysed bacteria expressing mCP α1β2 (lane 1), the flow through from the GST-mCAH3 Sepharose affinity column (lane 2), the low pH eluate from the GST-mCAH3 resin (lane 3), and the pooled fractions from Mono Q (lane 4). M, molecular weight standards.
Figure Legend Snippet: Large scale purification of mouse CP from bacterial lysates using GST-mCAH3 as an affinity matrix Samples taken during the purification procedure were separated by SDS-PAGE and stained with Coomassie Blue. Shown are the HSS of lysed bacteria expressing mCP α1β2 (lane 1), the flow through from the GST-mCAH3 Sepharose affinity column (lane 2), the low pH eluate from the GST-mCAH3 resin (lane 3), and the pooled fractions from Mono Q (lane 4). M, molecular weight standards.

Techniques Used: Purification, SDS Page, Staining, Expressing, Flow Cytometry, Affinity Column, Molecular Weight

28) Product Images from "Purification of Capping Protein Using the Capping Protein Binding Site of CARMIL as an Affinity Matrix"

Article Title: Purification of Capping Protein Using the Capping Protein Binding Site of CARMIL as an Affinity Matrix

Journal: Protein expression and purification

doi: 10.1016/j.pep.2009.05.002

Quantitative immunoblot analyses of the distributions of CP in ammonium sulfate fractions and small scale CP purifications using Acanthamoeba HSS The HSS of lysed Acanthamoeba was subjected to ammonium sulfate fractionation (Panel A: 0–25% pellet, 25–55% pellet and 55% supernatant in lanes 2, 4 and 3, respectively), a single ammonium sulfate precipitation (Panel B: 0–55% supernatant and 0–55% pellet in lanes 2 and 3, respectively), or not fractionated (Panel C). The 0–55% supernatant in Panel B was then subjected to small scale affinity chromatography on GST-AP Sepharose, and the unbound and bound fractions obtained (Panel B, lanes 4 and 5, respectively). Similarly, the HSS in Panel C was subjected to small scale affinity chromatography on GST-AP Sepharose, and the unbound and bound fractions obtained (Panel C, lanes 2 and 3, respectively). All of the samples in Panels AC were then probed by Western blotting using a polyclonal antibody raised against the α-subunit of Acanthamoeba ]. To allow the estimation of the relative distribution of CP in various ammonium sulfate fractions and in bound versus unbound fractions, the percent of the total fraction loaded was the same for the samples in lanes 2–4 in Panel A, for the samples in lanes 2–3 in Panel B, for the samples in lanes 4 and 5 in Panel B, and for the samples in lanes 2 and 3 in Panel C.
Figure Legend Snippet: Quantitative immunoblot analyses of the distributions of CP in ammonium sulfate fractions and small scale CP purifications using Acanthamoeba HSS The HSS of lysed Acanthamoeba was subjected to ammonium sulfate fractionation (Panel A: 0–25% pellet, 25–55% pellet and 55% supernatant in lanes 2, 4 and 3, respectively), a single ammonium sulfate precipitation (Panel B: 0–55% supernatant and 0–55% pellet in lanes 2 and 3, respectively), or not fractionated (Panel C). The 0–55% supernatant in Panel B was then subjected to small scale affinity chromatography on GST-AP Sepharose, and the unbound and bound fractions obtained (Panel B, lanes 4 and 5, respectively). Similarly, the HSS in Panel C was subjected to small scale affinity chromatography on GST-AP Sepharose, and the unbound and bound fractions obtained (Panel C, lanes 2 and 3, respectively). All of the samples in Panels AC were then probed by Western blotting using a polyclonal antibody raised against the α-subunit of Acanthamoeba ]. To allow the estimation of the relative distribution of CP in various ammonium sulfate fractions and in bound versus unbound fractions, the percent of the total fraction loaded was the same for the samples in lanes 2–4 in Panel A, for the samples in lanes 2–3 in Panel B, for the samples in lanes 4 and 5 in Panel B, and for the samples in lanes 2 and 3 in Panel C.

Techniques Used: Fractionation, Affinity Chromatography, Western Blot

Generation of the GST-AP affinity matrix Fractions sampled during the procedure were resolved by SDS-PAGE and stained with Coomassie Blue. Shown are lysates of E. coli harboring the GST-AP plasmid before (lane 1) and 3 h after induction of protein expression (lane 2), as well as the HSS (i.e. the soluble fraction) (lane 3) that was then captured on Glutathione Sepharose beads. After elution of GST-AP (lane 4) and dialysis against coupling buffer, the fusion protein was immobilized on CNBr-activated Sepharose. Coupling appeared to be complete, as the flow through from the coupling reaction (lane 5) was devoid of GST-AP. M, molecular mass standards.
Figure Legend Snippet: Generation of the GST-AP affinity matrix Fractions sampled during the procedure were resolved by SDS-PAGE and stained with Coomassie Blue. Shown are lysates of E. coli harboring the GST-AP plasmid before (lane 1) and 3 h after induction of protein expression (lane 2), as well as the HSS (i.e. the soluble fraction) (lane 3) that was then captured on Glutathione Sepharose beads. After elution of GST-AP (lane 4) and dialysis against coupling buffer, the fusion protein was immobilized on CNBr-activated Sepharose. Coupling appeared to be complete, as the flow through from the coupling reaction (lane 5) was devoid of GST-AP. M, molecular mass standards.

Techniques Used: SDS Page, Staining, Plasmid Preparation, Expressing, Flow Cytometry

Large scale purification of mouse CP from bacterial lysates using GST-mCAH3 as an affinity matrix Samples taken during the purification procedure were separated by SDS-PAGE and stained with Coomassie Blue. Shown are the HSS of lysed bacteria expressing mCP α1β2 (lane 1), the flow through from the GST-mCAH3 Sepharose affinity column (lane 2), the low pH eluate from the GST-mCAH3 resin (lane 3), and the pooled fractions from Mono Q (lane 4). M, molecular weight standards.
Figure Legend Snippet: Large scale purification of mouse CP from bacterial lysates using GST-mCAH3 as an affinity matrix Samples taken during the purification procedure were separated by SDS-PAGE and stained with Coomassie Blue. Shown are the HSS of lysed bacteria expressing mCP α1β2 (lane 1), the flow through from the GST-mCAH3 Sepharose affinity column (lane 2), the low pH eluate from the GST-mCAH3 resin (lane 3), and the pooled fractions from Mono Q (lane 4). M, molecular weight standards.

Techniques Used: Purification, SDS Page, Staining, Expressing, Flow Cytometry, Affinity Column, Molecular Weight

29) Product Images from "Saccharomyces cerevisiae Bzz1p Is Implicated with Type I Myosins in Actin Patch Polarization and Is Able To Recruit Actin-Polymerizing Machinery In Vitro"

Article Title: Saccharomyces cerevisiae Bzz1p Is Implicated with Type I Myosins in Actin Patch Polarization and Is Able To Recruit Actin-Polymerizing Machinery In Vitro

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.22.7889-7906.2002

Bzz1p recruits the actin polymerization machinery in vitro. (A) Glutathione-Sepharose beads coated with either GST or GST fused to full-length Bzz1p or the two C-terminal SH3 domains of Bzz1p were incubated with extracts from either the bzz1 Δ cells (FSW701K, upper panel) or the bzz1 Δ las17 Δ cells (FSW717KH middle panel) in the presence of Alexa-actin. Samples were incubated at room temperature, and the fluorescent signal was visualized. The photos shown below indicate representative positive (+) and negative (−) signals. In the lower panel ( bzz1 Δ las17 Δ + 6His-Las17p), GST fusion protein-coated beads were first incubated with an excess of purified 6His-Las17p (see Materials and Methods) for 20 min. After extensive washing, the beads were tested for the actin polymerization assay by using the lsb7 Δ las17 Δ-derived extract. Panel B summarizes the results obtained and shows the results from experiments with extracts from wild-type ( WT [SLW001]), arp2 - 2 (FKW201), bzz1 Δ vrp1 Δ (FSW751KK), bzz1 Δ myo5 Δ (SLW571KT), bzz1 Δ myo3 Δ (SLW371HK), and myo5 Δ myo3 Δ (RLY822) strains. Latrunculin A (LatA) was added to the bzz1 Δ + LatA sample at the 0-h incubation. For the bzz1 Δ + Pha + LatA sample, actin filament polymerized in the absence of beads was stabilized with phalloidin (Pha) prior to the addition of GST-Bzz1p-coated beads and latrunculin A.
Figure Legend Snippet: Bzz1p recruits the actin polymerization machinery in vitro. (A) Glutathione-Sepharose beads coated with either GST or GST fused to full-length Bzz1p or the two C-terminal SH3 domains of Bzz1p were incubated with extracts from either the bzz1 Δ cells (FSW701K, upper panel) or the bzz1 Δ las17 Δ cells (FSW717KH middle panel) in the presence of Alexa-actin. Samples were incubated at room temperature, and the fluorescent signal was visualized. The photos shown below indicate representative positive (+) and negative (−) signals. In the lower panel ( bzz1 Δ las17 Δ + 6His-Las17p), GST fusion protein-coated beads were first incubated with an excess of purified 6His-Las17p (see Materials and Methods) for 20 min. After extensive washing, the beads were tested for the actin polymerization assay by using the lsb7 Δ las17 Δ-derived extract. Panel B summarizes the results obtained and shows the results from experiments with extracts from wild-type ( WT [SLW001]), arp2 - 2 (FKW201), bzz1 Δ vrp1 Δ (FSW751KK), bzz1 Δ myo5 Δ (SLW571KT), bzz1 Δ myo3 Δ (SLW371HK), and myo5 Δ myo3 Δ (RLY822) strains. Latrunculin A (LatA) was added to the bzz1 Δ + LatA sample at the 0-h incubation. For the bzz1 Δ + Pha + LatA sample, actin filament polymerized in the absence of beads was stabilized with phalloidin (Pha) prior to the addition of GST-Bzz1p-coated beads and latrunculin A.

Techniques Used: In Vitro, Incubation, Purification, Polymerization Assay, Derivative Assay

Las17p can also recruit the actin polymerization machinery in vitro . (A) Ni-NTA-agarose beads coated with six His fused to full-length Las17p were incubated in the presence of small amounts of Alexa-labeled actin with extracts from either the wild-type ( WT ) strain (CLW001), the bzz1 Δ strain (FSW701K), the bzz1 Δ myo5 Δ strain (CLW571KT), or the myo3 Δ myo5 Δ strain (RLY822) or without yeast extract. After 10 to 20 min of incubation at room temperature, the fluorescence signal was visualized. The photos shown below indicate representative positive (+) and negative (−) signals. Panel B summarizes the results obtained.
Figure Legend Snippet: Las17p can also recruit the actin polymerization machinery in vitro . (A) Ni-NTA-agarose beads coated with six His fused to full-length Las17p were incubated in the presence of small amounts of Alexa-labeled actin with extracts from either the wild-type ( WT ) strain (CLW001), the bzz1 Δ strain (FSW701K), the bzz1 Δ myo5 Δ strain (CLW571KT), or the myo3 Δ myo5 Δ strain (RLY822) or without yeast extract. After 10 to 20 min of incubation at room temperature, the fluorescence signal was visualized. The photos shown below indicate representative positive (+) and negative (−) signals. Panel B summarizes the results obtained.

Techniques Used: In Vitro, Incubation, Labeling, Fluorescence

Las17p interacts with Bzz1p in vivo and in vitro. (A) Two-hybrid interaction between Las17p and portions of Bzz1p. pACTII vectors carrying different GAL4-AD-BZZ1 gene fusions were transformed into Y190 cells expressing GAL4 - DB in fusion with full-length LAS17 . For each combination, extracts of three transformants were assayed for β-galactosidase activity. Interactions stronger than those of negative controls are shown as “+.” Empty vectors were used as negative controls, and the DB- ACT1 AD- PFY1 vector was used as a positive control. DB, DNA-binding domain; AD, activation domain. (B) Bzz1p interacts directly with Las17p in vitro. GST fused to full-length Bzz1p (GST-Bzz1p), GST fused to the COOH-terminal 188 aa of Bzz1p containing the two SH3 domains (GST-SH3), and six His fused to full-length Las17p (6His-Las17p) were purified from bacteria (see Materials and Methods). The same amount of GST fusion proteins (GST-Bzz1p or GST-SH3SH3) or GST alone bound to glutathione-Sepharose beads was incubated with a fixed amount of purified 6His-Las17p. After washing, total (T), bound (B), and unbound (UB) fractions were analyzed by Western blotting with Ni-NTA-HRP conjugate to reveal the 6His-Las17p fusion protein. (C) Two-hybrid interactions between Bzz1p and fragments of Las17p. pASΔΔ vectors containing different segments of LAS17 fused with GAL4-DB were transformed into Y190 cells expressing GAL4-AD in fusion with full-length BZZ1 . Activity was assayed as in panel A.
Figure Legend Snippet: Las17p interacts with Bzz1p in vivo and in vitro. (A) Two-hybrid interaction between Las17p and portions of Bzz1p. pACTII vectors carrying different GAL4-AD-BZZ1 gene fusions were transformed into Y190 cells expressing GAL4 - DB in fusion with full-length LAS17 . For each combination, extracts of three transformants were assayed for β-galactosidase activity. Interactions stronger than those of negative controls are shown as “+.” Empty vectors were used as negative controls, and the DB- ACT1 AD- PFY1 vector was used as a positive control. DB, DNA-binding domain; AD, activation domain. (B) Bzz1p interacts directly with Las17p in vitro. GST fused to full-length Bzz1p (GST-Bzz1p), GST fused to the COOH-terminal 188 aa of Bzz1p containing the two SH3 domains (GST-SH3), and six His fused to full-length Las17p (6His-Las17p) were purified from bacteria (see Materials and Methods). The same amount of GST fusion proteins (GST-Bzz1p or GST-SH3SH3) or GST alone bound to glutathione-Sepharose beads was incubated with a fixed amount of purified 6His-Las17p. After washing, total (T), bound (B), and unbound (UB) fractions were analyzed by Western blotting with Ni-NTA-HRP conjugate to reveal the 6His-Las17p fusion protein. (C) Two-hybrid interactions between Bzz1p and fragments of Las17p. pASΔΔ vectors containing different segments of LAS17 fused with GAL4-DB were transformed into Y190 cells expressing GAL4-AD in fusion with full-length BZZ1 . Activity was assayed as in panel A.

Techniques Used: In Vivo, In Vitro, Transformation Assay, Expressing, Activity Assay, Plasmid Preparation, Positive Control, Binding Assay, Activation Assay, Purification, Incubation, Western Blot

30) Product Images from "RACK1 is involved in endothelial barrier regulation via its two novel interacting partners"

Article Title: RACK1 is involved in endothelial barrier regulation via its two novel interacting partners

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/1478-811X-11-2

Domain mapping of TIMAP-RACK1 interaction. ( A ): GST-TIMAP pull-down of endogenous RACK1. GST, recombinant GST-TIMAP WT or additional GST-TIMAP fragments (depicted in the upper part of panel A ) were loaded onto glutathione-Sepharose as described in Materials and Methods. The immobilized protein samples were incubated with BPAEC lysate. Western blot of the pull-down eluates probed with anti-RACK1 antibody is shown. CL: total cell lysate. ( B ): GST-RACK1 pull-down of endogenous TIMAP. GST, recombinant RACK1 WT or GST-RACK1 fragments (depicted in the upper part of panel B ) were tested in pull-down assay. The immobilized samples were incubated with BPAEC lysate. Western blot of the pull-down eluates was probed with anti-TIMAP antibody. Representative data of at least 3 independent experiments are shown.
Figure Legend Snippet: Domain mapping of TIMAP-RACK1 interaction. ( A ): GST-TIMAP pull-down of endogenous RACK1. GST, recombinant GST-TIMAP WT or additional GST-TIMAP fragments (depicted in the upper part of panel A ) were loaded onto glutathione-Sepharose as described in Materials and Methods. The immobilized protein samples were incubated with BPAEC lysate. Western blot of the pull-down eluates probed with anti-RACK1 antibody is shown. CL: total cell lysate. ( B ): GST-RACK1 pull-down of endogenous TIMAP. GST, recombinant RACK1 WT or GST-RACK1 fragments (depicted in the upper part of panel B ) were tested in pull-down assay. The immobilized samples were incubated with BPAEC lysate. Western blot of the pull-down eluates was probed with anti-TIMAP antibody. Representative data of at least 3 independent experiments are shown.

Techniques Used: Recombinant, Incubation, Western Blot, Pull Down Assay

TIMAP-RACK1 interaction is attenuated by the cAMP/PKA pathway. ( A ) GST, full-length GST-TIMAP (upper part) or GST-RACK1 (lower part) were immobilized on glutathione-Sepharose and incubated with cell lysates of non treated (ctr), forskolin (50 μM for 30 min) (FRSK) or PMA (1 μM for 30 min) treated BPAEC. The eluted proteins were tested by Western blot using anti-RACK1 and anti-TIMAP antibodies. ( B ) Endogenous TIMAP or RACK1 was immunoprecipitated from BPAEC lysates after the same treatments described for panel A . IP complexes were probed for TIMAP and RACK1. Shown are representative data of means ± SE from at least 3 independent experiments. Protein levels were quantified by densitometric analysis. Eluted proteins were normalized against total protein levels.
Figure Legend Snippet: TIMAP-RACK1 interaction is attenuated by the cAMP/PKA pathway. ( A ) GST, full-length GST-TIMAP (upper part) or GST-RACK1 (lower part) were immobilized on glutathione-Sepharose and incubated with cell lysates of non treated (ctr), forskolin (50 μM for 30 min) (FRSK) or PMA (1 μM for 30 min) treated BPAEC. The eluted proteins were tested by Western blot using anti-RACK1 and anti-TIMAP antibodies. ( B ) Endogenous TIMAP or RACK1 was immunoprecipitated from BPAEC lysates after the same treatments described for panel A . IP complexes were probed for TIMAP and RACK1. Shown are representative data of means ± SE from at least 3 independent experiments. Protein levels were quantified by densitometric analysis. Eluted proteins were normalized against total protein levels.

Techniques Used: Incubation, Western Blot, Immunoprecipitation

RACK1 interacts with PP1cδ via TIMAP. ( A ): Bacterially expressed glutathione S-transferase (GST) and GST-tagged wild-type TIMAP were loaded onto glutathione-Sepharose as described in Materials and Methods. After a washing step the resin samples were incubated with BPAEC lysate (CL) or cell lysis buffer (LB). Non-binding proteins were washed out and the bound proteins were eluted with 10 mM glutathion. Western blot probed with RACK1 specific antibody ( A ) of the endothelial cell lysate (CL) and the eluted fractions after the pull-down are shown. ( B , C ): RACK1 or TIMAP was immunoprecipitated from lysates of BPAEC ( B ) and BPAEC or HeLa ( C ) cells as described in Materials and Methods. IP complexes were probed for TIMAP and RACK1 ( B ) or PP1cδ ( C ). CL: cell lysate, Ø AB: control of IP from BPAEC without the addition of antibody. ( D , E ): RACK1 or GFP was immunoprecipitated from non transfected (CL), non-siRNA, TIMAP specific siRNA (si TIMAP), pEGFP-C1 (GFP), pEGFP-C1 TIMAP WT (GFP-Twt) or pEGFP-C1 TIMAPΔpp1c (GFP-TΔ) transfected HeLa cell lysates. IP complexes were probed for GFP, RACK1 or PP1cδ.
Figure Legend Snippet: RACK1 interacts with PP1cδ via TIMAP. ( A ): Bacterially expressed glutathione S-transferase (GST) and GST-tagged wild-type TIMAP were loaded onto glutathione-Sepharose as described in Materials and Methods. After a washing step the resin samples were incubated with BPAEC lysate (CL) or cell lysis buffer (LB). Non-binding proteins were washed out and the bound proteins were eluted with 10 mM glutathion. Western blot probed with RACK1 specific antibody ( A ) of the endothelial cell lysate (CL) and the eluted fractions after the pull-down are shown. ( B , C ): RACK1 or TIMAP was immunoprecipitated from lysates of BPAEC ( B ) and BPAEC or HeLa ( C ) cells as described in Materials and Methods. IP complexes were probed for TIMAP and RACK1 ( B ) or PP1cδ ( C ). CL: cell lysate, Ø AB: control of IP from BPAEC without the addition of antibody. ( D , E ): RACK1 or GFP was immunoprecipitated from non transfected (CL), non-siRNA, TIMAP specific siRNA (si TIMAP), pEGFP-C1 (GFP), pEGFP-C1 TIMAP WT (GFP-Twt) or pEGFP-C1 TIMAPΔpp1c (GFP-TΔ) transfected HeLa cell lysates. IP complexes were probed for GFP, RACK1 or PP1cδ.

Techniques Used: Incubation, Lysis, Binding Assay, Western Blot, Immunoprecipitation, Transfection

31) Product Images from "p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage"

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

Journal: Nature Communications

doi: 10.1038/s41467-018-03417-3

NELFE phosphorylation on S115 is required for the interaction with 14-3-3. a Schematic representation of NELFE domain organization and phosphorylation sites that were identified by phosphoproteomics. The SILAC ratios quantified for phosphorylation sites on NELFE after UV light and p38 inhibition are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. b The table shows all phosphorylation sites identified on NELFE by phosphoproteomics. The position, SILAC ratios, 14-3-3 binding prediction and sequence window are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. c Serine 115 phosphorylation is required for the interaction of NELFE and 14-3-3. U2OS cells expressing GFP-tagged wild-type NELFE or NELFE serine-to-alanine mutants were irradiated with UV light. Protein extracts were incubated with GST-14-3-3 and enriched proteins were resolved on SDS-PAGE. d Mass spectrometric parent ion scan of the peptide SISADDDLQESSR corresponding to S115 in NELFE. The SILAC triplet shows the relative abundance and mass to charge (m/z) of the phosphorylated peptide in mock-treated cells and cells irradiated with UV light without or with pretreatment with the p38 inhibitor. e Absolute occupancy of serine 49, 51, 115, and 251 phosphorylation in NELFE in undamaged cells and after UV light was determined by MS. f NELFE S115A mutant does not bind to 14-3-3. SILAC-labeled cells overexpressing GFP-tagged wild-type NELFE or NELFE S115A mutant were irradiated with UV light. UV-light-irradiated U2OS cells overexpressing GFP alone were used as control. Cells were lysed and protein extracts were incubated with GFP Trap agarose. The scatter plot shows the logarithmized SILAC ratios of quantified proteins. The color-coding indicates the density. g Recombinant 14-3-3 binds to phosphorylated NELFE peptide. Biotinylated phosphorylated NELFE peptide corresponding to serine 115 was bound to NeutrAvidin agarose. Phosphorylated and dephosphorylated peptide were incubated with purified 14-3-3. h Structure of 14-3-3 epsilon in complex with NELFE phosphorylated peptide QPFQRSI(p)SADDDLQE. Structure of the 14-3-3 epsilon in cartoon representation (Yellow and Cyan) and NELFE phosphorylated peptide in ball and stick model (Green). The inset on the right shows the 14-3-3 epsilon–NELFE phosphorylated peptide interaction
Figure Legend Snippet: NELFE phosphorylation on S115 is required for the interaction with 14-3-3. a Schematic representation of NELFE domain organization and phosphorylation sites that were identified by phosphoproteomics. The SILAC ratios quantified for phosphorylation sites on NELFE after UV light and p38 inhibition are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. b The table shows all phosphorylation sites identified on NELFE by phosphoproteomics. The position, SILAC ratios, 14-3-3 binding prediction and sequence window are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. c Serine 115 phosphorylation is required for the interaction of NELFE and 14-3-3. U2OS cells expressing GFP-tagged wild-type NELFE or NELFE serine-to-alanine mutants were irradiated with UV light. Protein extracts were incubated with GST-14-3-3 and enriched proteins were resolved on SDS-PAGE. d Mass spectrometric parent ion scan of the peptide SISADDDLQESSR corresponding to S115 in NELFE. The SILAC triplet shows the relative abundance and mass to charge (m/z) of the phosphorylated peptide in mock-treated cells and cells irradiated with UV light without or with pretreatment with the p38 inhibitor. e Absolute occupancy of serine 49, 51, 115, and 251 phosphorylation in NELFE in undamaged cells and after UV light was determined by MS. f NELFE S115A mutant does not bind to 14-3-3. SILAC-labeled cells overexpressing GFP-tagged wild-type NELFE or NELFE S115A mutant were irradiated with UV light. UV-light-irradiated U2OS cells overexpressing GFP alone were used as control. Cells were lysed and protein extracts were incubated with GFP Trap agarose. The scatter plot shows the logarithmized SILAC ratios of quantified proteins. The color-coding indicates the density. g Recombinant 14-3-3 binds to phosphorylated NELFE peptide. Biotinylated phosphorylated NELFE peptide corresponding to serine 115 was bound to NeutrAvidin agarose. Phosphorylated and dephosphorylated peptide were incubated with purified 14-3-3. h Structure of 14-3-3 epsilon in complex with NELFE phosphorylated peptide QPFQRSI(p)SADDDLQE. Structure of the 14-3-3 epsilon in cartoon representation (Yellow and Cyan) and NELFE phosphorylated peptide in ball and stick model (Green). The inset on the right shows the 14-3-3 epsilon–NELFE phosphorylated peptide interaction

Techniques Used: Inhibition, Labeling, Binding Assay, Sequencing, Expressing, Irradiation, Incubation, SDS Page, Mass Spectrometry, Mutagenesis, Recombinant, Purification

UV-light-induced phosphorylation of NELFE by MK2 leads to 14-3-3 binding. a Identification of p38-dependent NELFE interaction partners after UV light. SILAC-labeled U2OS cells expressing GFP-NELFE were mock-treated or irradiated with UV light. Cells were lysed and protein extracts were incubated with GFP Trap agarose. Enriched proteins were resolved on SDS-PAGE and digested in-gel into peptides. Peptides were extracted from gel and analyzed by LC-MS/MS. The scatter plot shows the logarithmized SILAC ratios of proteins quantified in the pull down. The color coding indicates the density. b NELFE interaction with 14-3-3 after UV light is p38- and MK2/3/5-dependent. U2OS cells expressing Flag-Strep-14-3-3 or an empty vector were mock-treated, irradiated with UV light or pretreated with the p38 or MK2/3/5 inhibitor, and then irradiated with UV light. Cells were lysed and protein extracts were incubated with StrepTactin sepharose. Enriched proteins were resolved by SDS-PAGE and selected proteins were detected with the indicated antibodies. c NELFE interaction with GST-14-3-3 is abolished in p38 and MK2 knockdown cells. U2OS cells were transfected with non-targeting, p38, or MK2-targeting siRNA and then irradiated with UV light. Cells were lysed and protein extracts were incubated with recombinant GST-14-3-3. Enriched proteins were resolved by SDS-PAGE and NELFE was detected using a specific antibody. d NELFE is phosphorylated after UV light on a 14-3-3-binding motif. GFP-NELFE was pulled down using GFP Trap agarose. Phosphorylation of NELFE was detected using antibodies recognizing the 14-3-3 motif. NELFE knockdown was used as control. e NELFE interacts with 14-3-3 after inhibition of P-TEFb. U2OS cells were treated with the p38 inhibitor or P-TEFb inhibitor 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB) and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. f Dynamics of NELFE interaction with 14-3-3 after UV light. U2OS cells were exposed to UV light and left to recover for the indicated time points. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. g NELFE interaction with 14-3-3 is partially dependent on the NER machinery. U2OS cells were transfected with a non-targeting siRNA or siRNA targeting XPC or CSB and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3
Figure Legend Snippet: UV-light-induced phosphorylation of NELFE by MK2 leads to 14-3-3 binding. a Identification of p38-dependent NELFE interaction partners after UV light. SILAC-labeled U2OS cells expressing GFP-NELFE were mock-treated or irradiated with UV light. Cells were lysed and protein extracts were incubated with GFP Trap agarose. Enriched proteins were resolved on SDS-PAGE and digested in-gel into peptides. Peptides were extracted from gel and analyzed by LC-MS/MS. The scatter plot shows the logarithmized SILAC ratios of proteins quantified in the pull down. The color coding indicates the density. b NELFE interaction with 14-3-3 after UV light is p38- and MK2/3/5-dependent. U2OS cells expressing Flag-Strep-14-3-3 or an empty vector were mock-treated, irradiated with UV light or pretreated with the p38 or MK2/3/5 inhibitor, and then irradiated with UV light. Cells were lysed and protein extracts were incubated with StrepTactin sepharose. Enriched proteins were resolved by SDS-PAGE and selected proteins were detected with the indicated antibodies. c NELFE interaction with GST-14-3-3 is abolished in p38 and MK2 knockdown cells. U2OS cells were transfected with non-targeting, p38, or MK2-targeting siRNA and then irradiated with UV light. Cells were lysed and protein extracts were incubated with recombinant GST-14-3-3. Enriched proteins were resolved by SDS-PAGE and NELFE was detected using a specific antibody. d NELFE is phosphorylated after UV light on a 14-3-3-binding motif. GFP-NELFE was pulled down using GFP Trap agarose. Phosphorylation of NELFE was detected using antibodies recognizing the 14-3-3 motif. NELFE knockdown was used as control. e NELFE interacts with 14-3-3 after inhibition of P-TEFb. U2OS cells were treated with the p38 inhibitor or P-TEFb inhibitor 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB) and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. f Dynamics of NELFE interaction with 14-3-3 after UV light. U2OS cells were exposed to UV light and left to recover for the indicated time points. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. g NELFE interaction with 14-3-3 is partially dependent on the NER machinery. U2OS cells were transfected with a non-targeting siRNA or siRNA targeting XPC or CSB and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3

Techniques Used: Binding Assay, Labeling, Expressing, Irradiation, Incubation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation, Transfection, Recombinant, Inhibition, Lysis

32) Product Images from "F-Box Protein Specificity for G1 Cyclins Is Dictated by Subcellular Localization"

Article Title: F-Box Protein Specificity for G1 Cyclins Is Dictated by Subcellular Localization

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002851

Cdc4 can target Cln2 for degradation upon co-localization. (A) Cdk-phosphorylated Cln2 interacts with Cdc4 and Grr1. Myc and GST Western blots showing pull-down of GST, GST-Cdc4ΔF and GST-Grr1ΔF proteins from grr1Δ cells expressing Cln2-13Myc (wt) or Cln2-4T3S-13Myc (4T3S). 2% input (i) and glutathione-sepharose bound proteins (b) are shown. (B) Expression of cytoplasmic Cdc4 downregulates Cdk-phosphorylated Cln2. Western blot showing levels of Cln2-13Myc, or Cln2-4T3S-13Myc, in grr1Δ cells after induction of GST, GST-Cdc4-FLAG, or GST-NES-Cdc4-FLAG expression following the addition of galactose for the indicated number of hours. Levels of FLAG-tagged proteins and Cdc28 are also shown. For all gels, molecular-weight markers are indicated at the left.
Figure Legend Snippet: Cdc4 can target Cln2 for degradation upon co-localization. (A) Cdk-phosphorylated Cln2 interacts with Cdc4 and Grr1. Myc and GST Western blots showing pull-down of GST, GST-Cdc4ΔF and GST-Grr1ΔF proteins from grr1Δ cells expressing Cln2-13Myc (wt) or Cln2-4T3S-13Myc (4T3S). 2% input (i) and glutathione-sepharose bound proteins (b) are shown. (B) Expression of cytoplasmic Cdc4 downregulates Cdk-phosphorylated Cln2. Western blot showing levels of Cln2-13Myc, or Cln2-4T3S-13Myc, in grr1Δ cells after induction of GST, GST-Cdc4-FLAG, or GST-NES-Cdc4-FLAG expression following the addition of galactose for the indicated number of hours. Levels of FLAG-tagged proteins and Cdc28 are also shown. For all gels, molecular-weight markers are indicated at the left.

Techniques Used: Western Blot, Expressing, Molecular Weight

Cdk-phosphorylation of the Cln3 C-terminus is required for its degradation. (A) Mutation of Cdk consensus sites stabilizes Cln3. Cycloheximide-chase assay showing levels of Cln3-13Myc, Cln3-5A-13Myc, Cln3-3A-13Myc and Cln3-9A-13Myc after the addition of cycloheximide for the indicated number of minutes (min chx). Cdc28 is shown as a loading control. Diagram of mutant alleles is shown in Figure S2B . (B) Expression of Cln3-9A accelerates progression through G1 phase. Cell cycle profiles of asynchronous wild-type cells and cells expressing Cln3-3A, Cln3-5A, or Cln3-9A. (C) Cdc4 and Grr1 bind to Cdk-phosphorylated Cln3. Myc and GST Western blots showing pull-downs of GST, GST-Cdc4ΔF and GST-Grr1ΔF proteins from grr1Δ cdc4-1 cells expressing Cln3-13Myc (wt) or Cln3-9A-13Myc (9A). 2% input (i) and glutathione-sepharose bound proteins (b) are shown. (D) Cdc4 and Grr1 require different regions of the Cln3 C-terminus for targeting. Cycloheximide-chase assays of Cln3-5A-13Myc (top panels) and Cln3-3A-13Myc (bottom panels) in wild-type (wt), grr1Δ or cdc4-1 cells. Cells were shifted to 37°C for 2 hours and cycloheximide was added for the indicated number of minutes. Cln3-13Myc and Cdc28 Western blots are shown. (E) Cdk-phosphorylation of Cln3 occurs in cis . Cycloheximide-chase assay of full-length Cln3-13Myc, Cln3ΔN-13Myc (Cln3 lacking amino acids 2-207), and Cln3ΔN-9A-13Myc. Each protein is expressed from the TEF1 promoter. For all gels, molecular weight markers are indicated at the left.
Figure Legend Snippet: Cdk-phosphorylation of the Cln3 C-terminus is required for its degradation. (A) Mutation of Cdk consensus sites stabilizes Cln3. Cycloheximide-chase assay showing levels of Cln3-13Myc, Cln3-5A-13Myc, Cln3-3A-13Myc and Cln3-9A-13Myc after the addition of cycloheximide for the indicated number of minutes (min chx). Cdc28 is shown as a loading control. Diagram of mutant alleles is shown in Figure S2B . (B) Expression of Cln3-9A accelerates progression through G1 phase. Cell cycle profiles of asynchronous wild-type cells and cells expressing Cln3-3A, Cln3-5A, or Cln3-9A. (C) Cdc4 and Grr1 bind to Cdk-phosphorylated Cln3. Myc and GST Western blots showing pull-downs of GST, GST-Cdc4ΔF and GST-Grr1ΔF proteins from grr1Δ cdc4-1 cells expressing Cln3-13Myc (wt) or Cln3-9A-13Myc (9A). 2% input (i) and glutathione-sepharose bound proteins (b) are shown. (D) Cdc4 and Grr1 require different regions of the Cln3 C-terminus for targeting. Cycloheximide-chase assays of Cln3-5A-13Myc (top panels) and Cln3-3A-13Myc (bottom panels) in wild-type (wt), grr1Δ or cdc4-1 cells. Cells were shifted to 37°C for 2 hours and cycloheximide was added for the indicated number of minutes. Cln3-13Myc and Cdc28 Western blots are shown. (E) Cdk-phosphorylation of Cln3 occurs in cis . Cycloheximide-chase assay of full-length Cln3-13Myc, Cln3ΔN-13Myc (Cln3 lacking amino acids 2-207), and Cln3ΔN-9A-13Myc. Each protein is expressed from the TEF1 promoter. For all gels, molecular weight markers are indicated at the left.

Techniques Used: Mutagenesis, Expressing, Western Blot, Molecular Weight

Cln3 interacts with Cdc4 and Grr1. (A) Cln3 interacts with Cdc4 and Grr1. Myc and GST Western blots showing pull-downs of GST, GST-Cdc4ΔF and GST-Grr1ΔF proteins from grr1Δ cdc4-1 cells expressing Cln3-13Myc (wt) or Cln3 truncation mutants (described in Figure S2B ). 2% input (i) and glutathione-sepharose bound proteins (b) are shown. (B) Cycloheximide-chase assay of Cln3 truncation mutants from (A) expressed in wild-type cells. Western blots for Myc and Cdc28 are shown. For all gels, molecular weight markers are indicated at the left.
Figure Legend Snippet: Cln3 interacts with Cdc4 and Grr1. (A) Cln3 interacts with Cdc4 and Grr1. Myc and GST Western blots showing pull-downs of GST, GST-Cdc4ΔF and GST-Grr1ΔF proteins from grr1Δ cdc4-1 cells expressing Cln3-13Myc (wt) or Cln3 truncation mutants (described in Figure S2B ). 2% input (i) and glutathione-sepharose bound proteins (b) are shown. (B) Cycloheximide-chase assay of Cln3 truncation mutants from (A) expressed in wild-type cells. Western blots for Myc and Cdc28 are shown. For all gels, molecular weight markers are indicated at the left.

Techniques Used: Western Blot, Expressing, Molecular Weight

33) Product Images from "Fbw7 Targets GATA3 through Cyclin-Dependent Kinase 2-Dependent Proteolysis and Contributes to Regulation of T-Cell Development"

Article Title: Fbw7 Targets GATA3 through Cyclin-Dependent Kinase 2-Dependent Proteolysis and Contributes to Regulation of T-Cell Development

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01549-13

Phosphorylation at Thr-156 in GATA3 by CDK2 is required for association of Fbw7 and is executed in HUT78 cells during G 2 /M phase and in thymocytes of mice. (A) Depletion of CDK2 reduced phosphorylation of Thr-156 in GATA3 in HEK293 cells. HEK293 cells were transfected with a WT GATA3 expression plasmid and CDK2 or a control siRNA. After 43 h of transfection, cells were treated with okadaic acid and MG132 for 5 h. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. The numbers reflect the ratio of the levels of the indicated proteins between the CDK2 siRNA-transfected cells and control cells. (B) GATA3 binding to Fbw7 in vitro is Thr-156 phosphorylation dependent. Purified WT or T156A GST-GATA3 was incubated with the indicated kinases in reaction buffer at 30°C for 30 min. Lysates from HEK293 cells transfected with HA-Fbw7 were immunoprecipitated with HA antibody, and the immunocomplexes containing Fbw7 were incubated with phosphorylated GST-GATA3 in vitro as indicated. To analyze GATA3 and Fbw7 binding, the GST fusion protein complexes were precipitated using glutathione-Sepharose beads and subjected to immunoblotting with the indicated antibodies. (C and D) Phosphorylation of recombinant GATA3 in vitro . Reaction products were then subjected to immunoblot analysis with the indicated antibodies. (C) Phosphorylation of Thr-156 in GATA3 in G 2 /M-arrested cells. GST-GATA3 was incubated with lysate prepared from either cells arrested in G 1 /S or G 2 /M phase or nonsynchronized (AS) HeLa cells. (D) Effects of CDK2 inhibition on Thr-156 phosphorylation of GATA3 in G 2 /M cell lysate. The responsible kinase in G 2 /M-phase cell lysate is CDK2. GST-GATA3 was incubated with the indicated kinase sources in the absence or presence of CDK2 inhibitor (CVT313) or competitor (p27). (E and F) Thr-156 phosphorylation of endogenous GATA3 in G 2 /M phase in T-cell lymphoma HUT78 cells. G 2 /M-arrested and asynchronous (AS) HUT78 cells were prepared as indicated in Materials and Methods. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. (F) G 2 /M-arrested and asynchronous cells were treated with or without MG132 for 5 h before harvest. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. (G) Cell lysate from whole thymocytes obtained from an Fbw7 flox/flox mouse at 6 weeks of age was incubated with or without calf intestinal alkaline phosphatase (CIAP) at 37°C for 30 min and subjected to immunoblot analysis with the indicated antibodies.
Figure Legend Snippet: Phosphorylation at Thr-156 in GATA3 by CDK2 is required for association of Fbw7 and is executed in HUT78 cells during G 2 /M phase and in thymocytes of mice. (A) Depletion of CDK2 reduced phosphorylation of Thr-156 in GATA3 in HEK293 cells. HEK293 cells were transfected with a WT GATA3 expression plasmid and CDK2 or a control siRNA. After 43 h of transfection, cells were treated with okadaic acid and MG132 for 5 h. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. The numbers reflect the ratio of the levels of the indicated proteins between the CDK2 siRNA-transfected cells and control cells. (B) GATA3 binding to Fbw7 in vitro is Thr-156 phosphorylation dependent. Purified WT or T156A GST-GATA3 was incubated with the indicated kinases in reaction buffer at 30°C for 30 min. Lysates from HEK293 cells transfected with HA-Fbw7 were immunoprecipitated with HA antibody, and the immunocomplexes containing Fbw7 were incubated with phosphorylated GST-GATA3 in vitro as indicated. To analyze GATA3 and Fbw7 binding, the GST fusion protein complexes were precipitated using glutathione-Sepharose beads and subjected to immunoblotting with the indicated antibodies. (C and D) Phosphorylation of recombinant GATA3 in vitro . Reaction products were then subjected to immunoblot analysis with the indicated antibodies. (C) Phosphorylation of Thr-156 in GATA3 in G 2 /M-arrested cells. GST-GATA3 was incubated with lysate prepared from either cells arrested in G 1 /S or G 2 /M phase or nonsynchronized (AS) HeLa cells. (D) Effects of CDK2 inhibition on Thr-156 phosphorylation of GATA3 in G 2 /M cell lysate. The responsible kinase in G 2 /M-phase cell lysate is CDK2. GST-GATA3 was incubated with the indicated kinase sources in the absence or presence of CDK2 inhibitor (CVT313) or competitor (p27). (E and F) Thr-156 phosphorylation of endogenous GATA3 in G 2 /M phase in T-cell lymphoma HUT78 cells. G 2 /M-arrested and asynchronous (AS) HUT78 cells were prepared as indicated in Materials and Methods. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. (F) G 2 /M-arrested and asynchronous cells were treated with or without MG132 for 5 h before harvest. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. (G) Cell lysate from whole thymocytes obtained from an Fbw7 flox/flox mouse at 6 weeks of age was incubated with or without calf intestinal alkaline phosphatase (CIAP) at 37°C for 30 min and subjected to immunoblot analysis with the indicated antibodies.

Techniques Used: Mouse Assay, Transfection, Expressing, Plasmid Preparation, Binding Assay, In Vitro, Purification, Incubation, Immunoprecipitation, Recombinant, Inhibition

Thr-156 of GATA3 is phosphorylated by CDK2. (A) Transiently expressed GATA3 is phosphorylated at Thr-156 in HEK293 cells. HEK293 cells were transfected with WT or T156A GATA3 and treated with 20 μM MG132 and 20 nM okadaic acid for 5 h to inhibit proteolysis and dephosphorylation of GATA3. Cell lysates were prepared with lysis buffer containing phosphatase inhibitors and protease inhibitors and subjected to immunoblot analysis using phospho-T156-GATA3 or Myc antibodies. (B) CDK2 phosphorylates GATA3 peptide in a Thr-156-dependent manner in vitro . A synthetic peptide corresponding to aa 150 to 161 of WT or T156A GATA3 was incubated with [γ- 32 ). To confirm the activities of the CDKs used in our experiments, we performed in vitro kinase assays using the S11 peptide as a positive control. Wild-type or T156A synthetic GATA3 peptide or S11 peptide was incubated with [γ- 32 P]ATP along with the indicated CDKs at 30°C for 30 min (bottom panel). The peptides were trapped on P81 papers and monitored for radioactivity using a liquid scintillation counter. (C and D) Recombinant GATA3 is phosphorylated at Thr-156 by CDK2 in vitro . WT or T156A GST-fused GATA3 was expressed in E. coli and affinity purified using glutathione-Sepharose 4B. GST alone was prepared in parallel as a control. The proteins were incubated with the kinases as indicated in reaction buffer at 30°C for 30 min. Reaction products were subjected to immunoblot analysis with the indicated antibodies.
Figure Legend Snippet: Thr-156 of GATA3 is phosphorylated by CDK2. (A) Transiently expressed GATA3 is phosphorylated at Thr-156 in HEK293 cells. HEK293 cells were transfected with WT or T156A GATA3 and treated with 20 μM MG132 and 20 nM okadaic acid for 5 h to inhibit proteolysis and dephosphorylation of GATA3. Cell lysates were prepared with lysis buffer containing phosphatase inhibitors and protease inhibitors and subjected to immunoblot analysis using phospho-T156-GATA3 or Myc antibodies. (B) CDK2 phosphorylates GATA3 peptide in a Thr-156-dependent manner in vitro . A synthetic peptide corresponding to aa 150 to 161 of WT or T156A GATA3 was incubated with [γ- 32 ). To confirm the activities of the CDKs used in our experiments, we performed in vitro kinase assays using the S11 peptide as a positive control. Wild-type or T156A synthetic GATA3 peptide or S11 peptide was incubated with [γ- 32 P]ATP along with the indicated CDKs at 30°C for 30 min (bottom panel). The peptides were trapped on P81 papers and monitored for radioactivity using a liquid scintillation counter. (C and D) Recombinant GATA3 is phosphorylated at Thr-156 by CDK2 in vitro . WT or T156A GST-fused GATA3 was expressed in E. coli and affinity purified using glutathione-Sepharose 4B. GST alone was prepared in parallel as a control. The proteins were incubated with the kinases as indicated in reaction buffer at 30°C for 30 min. Reaction products were subjected to immunoblot analysis with the indicated antibodies.

Techniques Used: Transfection, De-Phosphorylation Assay, Lysis, In Vitro, Incubation, Positive Control, Radioactivity, Recombinant, Affinity Purification

34) Product Images from "STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex"

Article Title: STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-017-0048-5

Effects of STAC2 on the RANK-Gab2-PLCγ2 complex. a The 293T cells were co-transfected with GST-RANK, PLCγ2, Gab2, and Flag-STAC2. Protein complexes were precipitated with Glutathione-Sepharose beads and subjected to western blot analysis using the indicated antibodies. b BMMs were infected with pMX-puro retrovirus (EV) and pMX-puro retrovirus expressing STAC2 (STAC2). The transduced BMMs were cultured with M-CSF and RANKL for 3 days and serum-starved. Cells were then stimulated with RANKL (300 ng/ml) for 15 min and lysed for immunoprecipitation with anti-RANK antibodies. Data are representative of at least three independent experiments a , b
Figure Legend Snippet: Effects of STAC2 on the RANK-Gab2-PLCγ2 complex. a The 293T cells were co-transfected with GST-RANK, PLCγ2, Gab2, and Flag-STAC2. Protein complexes were precipitated with Glutathione-Sepharose beads and subjected to western blot analysis using the indicated antibodies. b BMMs were infected with pMX-puro retrovirus (EV) and pMX-puro retrovirus expressing STAC2 (STAC2). The transduced BMMs were cultured with M-CSF and RANKL for 3 days and serum-starved. Cells were then stimulated with RANKL (300 ng/ml) for 15 min and lysed for immunoprecipitation with anti-RANK antibodies. Data are representative of at least three independent experiments a , b

Techniques Used: Transfection, Western Blot, Infection, Expressing, Cell Culture, Immunoprecipitation

STAC2 suppresses Btk/Tec-mediated PLCγ2 activation. a , b The 293T cells were transfected with PLCγ2, Flag-STAC2, and either Btk or Tec. Total whole-cell lysates were analyzed using the indicated antibodies. c , d The 293T cells were co-transfected with GST-Btk ( c ) or GST-Tec ( d ) together with PLCγ2 and Flag-STAC2 as indicated. Protein complexes were precipitated with Glutathione-Sepharose beads and subjected to western blot analysis using the corresponding antibodies. e BMMs transduced with retroviruses (EV and STAC2) were cultured with M-CSF and RANKL for 3 days. After serum starvation, BMMs were stimulated with RANKL (300 ng/ml) for 15 min. Cells were then lysed and immunoprecipitated with antibodies to Btk. Data are representative of at least three independent experiments a – d , or at least two independent experiments ( e )
Figure Legend Snippet: STAC2 suppresses Btk/Tec-mediated PLCγ2 activation. a , b The 293T cells were transfected with PLCγ2, Flag-STAC2, and either Btk or Tec. Total whole-cell lysates were analyzed using the indicated antibodies. c , d The 293T cells were co-transfected with GST-Btk ( c ) or GST-Tec ( d ) together with PLCγ2 and Flag-STAC2 as indicated. Protein complexes were precipitated with Glutathione-Sepharose beads and subjected to western blot analysis using the corresponding antibodies. e BMMs transduced with retroviruses (EV and STAC2) were cultured with M-CSF and RANKL for 3 days. After serum starvation, BMMs were stimulated with RANKL (300 ng/ml) for 15 min. Cells were then lysed and immunoprecipitated with antibodies to Btk. Data are representative of at least three independent experiments a – d , or at least two independent experiments ( e )

Techniques Used: Activation Assay, Transfection, Western Blot, Transduction, Cell Culture, Immunoprecipitation

STAC2 interacts with the IVVY motif of RANK. a The 293T cells were transfected with the indicated combinations of plasmids. Whole-cell lysates were prepared and subjected to immunoprecipitation with anti-Flag antibodies and analyzed by western blotting. b BMMs were cultured with M-CSF in the absence or presence of RANKL for 3 days. Cells were lysed and immunoprecipitated with anti-RANK antibodies. The resulting immunoprecipitated samples and total whole-cell lysates were subjected to western blot analysis with antibodies to RANK and STAC2. c Schematic diagrams of wild-type and mutant constructs of GST-RANK. Constructs that can interact with STAC2 are indicated by + . d The 293T cells were co-transfected with Flag-STAC2 and various GST-RANK mutants. Cell extracts were precipitated with Glutathione-Sepharose beads and subjected to western blot analysis using the indicated antibodies. Data are representative of at least three independent experiments ( a – d ). e Schematic diagrams of wild-type and deletion mutant constructs of Flag-STAC2. Constructs that can interact with RANK are indicated by + . f The 293T cells were co-transfected with GST-RANK and various Flag-STAC2 mutants. Cell extracts were precipitated with Glutathione-Sepharose beads and subjected to western blot analysis using the indicated antibodies. Data are representative of at least three independent experiments ( a – f )
Figure Legend Snippet: STAC2 interacts with the IVVY motif of RANK. a The 293T cells were transfected with the indicated combinations of plasmids. Whole-cell lysates were prepared and subjected to immunoprecipitation with anti-Flag antibodies and analyzed by western blotting. b BMMs were cultured with M-CSF in the absence or presence of RANKL for 3 days. Cells were lysed and immunoprecipitated with anti-RANK antibodies. The resulting immunoprecipitated samples and total whole-cell lysates were subjected to western blot analysis with antibodies to RANK and STAC2. c Schematic diagrams of wild-type and mutant constructs of GST-RANK. Constructs that can interact with STAC2 are indicated by + . d The 293T cells were co-transfected with Flag-STAC2 and various GST-RANK mutants. Cell extracts were precipitated with Glutathione-Sepharose beads and subjected to western blot analysis using the indicated antibodies. Data are representative of at least three independent experiments ( a – d ). e Schematic diagrams of wild-type and deletion mutant constructs of Flag-STAC2. Constructs that can interact with RANK are indicated by + . f The 293T cells were co-transfected with GST-RANK and various Flag-STAC2 mutants. Cell extracts were precipitated with Glutathione-Sepharose beads and subjected to western blot analysis using the indicated antibodies. Data are representative of at least three independent experiments ( a – f )

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

35) Product Images from "FIH-1: a novel protein that interacts with HIF-1? and VHL to mediate repression of HIF-1 transcriptional activity"

Article Title: FIH-1: a novel protein that interacts with HIF-1? and VHL to mediate repression of HIF-1 transcriptional activity

Journal: Genes & Development

doi: 10.1101/gad.924501

Interaction of VHL and FIH-1 with histone deacetylases. ( A ) GST and GST–HDAC fusion proteins were incubated with 35 S-labeled FLAG–VHL ( top ), HA–FIH-1 ( middle ), or HIF-1α ( bottom ), captured on glutathione–Sepharose beads, and analyzed by SDS-PAGE and autoradiography. ( B ) GST–HDAC fusion proteins were incubated with 35 S-labeled FLAG–VHL truncated at its C terminus as indicated and analyzed as described above. ( C ) GST–HDAC fusion proteins were incubated with 35 S-labeled FLAG–VHL and/or 35 S-labeled HIF-1α.
Figure Legend Snippet: Interaction of VHL and FIH-1 with histone deacetylases. ( A ) GST and GST–HDAC fusion proteins were incubated with 35 S-labeled FLAG–VHL ( top ), HA–FIH-1 ( middle ), or HIF-1α ( bottom ), captured on glutathione–Sepharose beads, and analyzed by SDS-PAGE and autoradiography. ( B ) GST–HDAC fusion proteins were incubated with 35 S-labeled FLAG–VHL truncated at its C terminus as indicated and analyzed as described above. ( C ) GST–HDAC fusion proteins were incubated with 35 S-labeled FLAG–VHL and/or 35 S-labeled HIF-1α.

Techniques Used: Incubation, Labeling, SDS Page, Autoradiography

Localization of HIF-1α residues interacting with FIH-1. ( A ) GST and GST–FIH-1 fusion proteins were expressed in E. coli , purified, incubated with 35 S-labeled in vitro-translated HIF-1α, captured on glutathione–Sepharose beads, and analyzed by SDS-PAGE and autoradiography. ( B – D ) GST-fusion proteins containing the indicated HIF-1α residues at their C terminus were incubated with 35 S-labeled in vitro-translated FIH-1, captured on glutathione–Sepharose beads, and analyzed as described above. (0) GST only.
Figure Legend Snippet: Localization of HIF-1α residues interacting with FIH-1. ( A ) GST and GST–FIH-1 fusion proteins were expressed in E. coli , purified, incubated with 35 S-labeled in vitro-translated HIF-1α, captured on glutathione–Sepharose beads, and analyzed by SDS-PAGE and autoradiography. ( B – D ) GST-fusion proteins containing the indicated HIF-1α residues at their C terminus were incubated with 35 S-labeled in vitro-translated FIH-1, captured on glutathione–Sepharose beads, and analyzed as described above. (0) GST only.

Techniques Used: Purification, Incubation, Labeling, In Vitro, SDS Page, Autoradiography

Interaction of FIH-1, HIF-1α, and VHL in vitro. ( A ) GST-fusion proteins containing HIF-1α residues 429–608 or 757–826 were expressed in E. coli , purified, and incubated with 35 S-labeled in vitro-translated FLAG-tagged VHL or HA-tagged FIH-1, captured on glutathione–Sepharose beads, and analyzed by SDS-PAGE and autoradiography. ( B ) 35 S-labeled in vitro-translated FIH-1 residues 1–349 or 126–349 was incubated with unlabeled FLAG–VHL ( top ) or GST–HIF-1α(531–826) ( middle ), which were pulled down on beads containing anti-FLAG antibody or glutathione, respectively, and analyzed by SDS-PAGE along with aliquots of the input FIH-1 polypeptides ( bottom ). ( C ) 35 S-labeled in vitro-translated FLAG–VHL truncated at its C terminus as indicated was incubated with unlabeled lysate-treated GST–HIF-1α(429–608) ( top ) or GST–HA–FIH-1 ( middle ), which were captured on glutathione–Sepharose beads and analyzed by SDS-PAGE along with aliquots of the input VHL polypeptides ( bottom ). ( D ) GST or the indicated GST–HIF-1α fusion protein was preincubated in reticulocyte lysate (odd-numbered lanes) or buffer (even-numbered lanes), incubated with 35 S-labeled FLAG–VHL, captured on glutathione–Sepharose beads, and analyzed as described above.
Figure Legend Snippet: Interaction of FIH-1, HIF-1α, and VHL in vitro. ( A ) GST-fusion proteins containing HIF-1α residues 429–608 or 757–826 were expressed in E. coli , purified, and incubated with 35 S-labeled in vitro-translated FLAG-tagged VHL or HA-tagged FIH-1, captured on glutathione–Sepharose beads, and analyzed by SDS-PAGE and autoradiography. ( B ) 35 S-labeled in vitro-translated FIH-1 residues 1–349 or 126–349 was incubated with unlabeled FLAG–VHL ( top ) or GST–HIF-1α(531–826) ( middle ), which were pulled down on beads containing anti-FLAG antibody or glutathione, respectively, and analyzed by SDS-PAGE along with aliquots of the input FIH-1 polypeptides ( bottom ). ( C ) 35 S-labeled in vitro-translated FLAG–VHL truncated at its C terminus as indicated was incubated with unlabeled lysate-treated GST–HIF-1α(429–608) ( top ) or GST–HA–FIH-1 ( middle ), which were captured on glutathione–Sepharose beads and analyzed by SDS-PAGE along with aliquots of the input VHL polypeptides ( bottom ). ( D ) GST or the indicated GST–HIF-1α fusion protein was preincubated in reticulocyte lysate (odd-numbered lanes) or buffer (even-numbered lanes), incubated with 35 S-labeled FLAG–VHL, captured on glutathione–Sepharose beads, and analyzed as described above.

Techniques Used: In Vitro, Purification, Incubation, Labeling, SDS Page, Autoradiography

36) Product Images from "Enigma Prevents Cbl-c-Mediated Ubiquitination and Degradation of RETMEN2A"

Article Title: Enigma Prevents Cbl-c-Mediated Ubiquitination and Degradation of RETMEN2A

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087116

Enigma interacts with Cbl-c. ( A ) HEK293T cells were transfected with GST, or GST-Cbl-c with and without untagged human Enigma as indicated above each blot. All transfections were balanced with empty vector controls and whole cell lysates were collected at 48 h post-transfection. GST pull-downs were performed using 300 µg of whole cell lysates. GST pull-downs (GST ppt.) and whole cell lysates (WCL) were immunoblotted (IB) as indicated to the right of each blot. Molecular weight standards in kDa are indicated to the left of each panel and Hsc70 is shown as a loading control. ( B ) HEK293T cells were transfected with HA-Cbl-c with and without FLAG-Enigma as indicated above each blot. Immunoprecipitations were performed using anti-FLAG-M2 agarose with 300 µg of whole cell lysates. IPs and WCL were immunoblotted as indicated to the right. Molecular weight standards are indicated to the left of each panel and Ponceau S staining serves as a loading control. ( C ) Immunoprecipitation of endogenous Cbl-c was performed on whole cell lysates from three human cell lines: 30mg of MB-231, a triple negative breast cancer cell line; 30mg of T47D, a human ER + breast cancer cell line; and 20mg of CFPac-1, a human pancreatic cancer cell line. Immunoprecipitations using rabbit anti-Cbl-c conjugated sepharose, were immunoblotted for endogenous Cbl-c (indicated by arrow) and for endogenous Enigma as indicated. Rabbit immunoglobulin is indicated with an asterisk (*) and molecular weight standards are indicated on the left of each panel. Whole cell lysates were immunoblotted for endogenous Enigma and Hsc70 which serves as a loading control. ( D ) HEK293T cells were transfected with HA-Cbl, Cbl-b, or Cbl-c with and without FLAG-Enigma. Cbl immunoprecipitations were performed on 300 µg of each of the whole cell lysates using anti-HA affinity matrix and immunoblotted for co-immunoprecipitation of Enigma, as indicated. Whole cell lysates were immunoblotted as indicated and molecular weight standards are indicated on the left of each panel. β-actin serves as a loading control. ( E ) Enigma truncation constructs used for mapping Cbl-c interaction site. ( F ) HEK293T cells were co-transfected with GST-Cbl-c along with FLAG-Enigma, ΔLIM3, ΔLIM2-3, or ΔLIM1-3. Whole cell lysates were collected and immunoblotted for Enigma constructs with anti-FLAG and for Cbl-c as indicated on the right. GST pull-downs were immunoblotted for GST-Cbl-c with anti-GST and for each of the FLAG-Enigma proteins as indicated. Arrow indicates expected position of ΔLIM1-3 protein. Molecular weight standards are indicated on the left of each panel and Ponceau S staining serves as a measure of protein loading.
Figure Legend Snippet: Enigma interacts with Cbl-c. ( A ) HEK293T cells were transfected with GST, or GST-Cbl-c with and without untagged human Enigma as indicated above each blot. All transfections were balanced with empty vector controls and whole cell lysates were collected at 48 h post-transfection. GST pull-downs were performed using 300 µg of whole cell lysates. GST pull-downs (GST ppt.) and whole cell lysates (WCL) were immunoblotted (IB) as indicated to the right of each blot. Molecular weight standards in kDa are indicated to the left of each panel and Hsc70 is shown as a loading control. ( B ) HEK293T cells were transfected with HA-Cbl-c with and without FLAG-Enigma as indicated above each blot. Immunoprecipitations were performed using anti-FLAG-M2 agarose with 300 µg of whole cell lysates. IPs and WCL were immunoblotted as indicated to the right. Molecular weight standards are indicated to the left of each panel and Ponceau S staining serves as a loading control. ( C ) Immunoprecipitation of endogenous Cbl-c was performed on whole cell lysates from three human cell lines: 30mg of MB-231, a triple negative breast cancer cell line; 30mg of T47D, a human ER + breast cancer cell line; and 20mg of CFPac-1, a human pancreatic cancer cell line. Immunoprecipitations using rabbit anti-Cbl-c conjugated sepharose, were immunoblotted for endogenous Cbl-c (indicated by arrow) and for endogenous Enigma as indicated. Rabbit immunoglobulin is indicated with an asterisk (*) and molecular weight standards are indicated on the left of each panel. Whole cell lysates were immunoblotted for endogenous Enigma and Hsc70 which serves as a loading control. ( D ) HEK293T cells were transfected with HA-Cbl, Cbl-b, or Cbl-c with and without FLAG-Enigma. Cbl immunoprecipitations were performed on 300 µg of each of the whole cell lysates using anti-HA affinity matrix and immunoblotted for co-immunoprecipitation of Enigma, as indicated. Whole cell lysates were immunoblotted as indicated and molecular weight standards are indicated on the left of each panel. β-actin serves as a loading control. ( E ) Enigma truncation constructs used for mapping Cbl-c interaction site. ( F ) HEK293T cells were co-transfected with GST-Cbl-c along with FLAG-Enigma, ΔLIM3, ΔLIM2-3, or ΔLIM1-3. Whole cell lysates were collected and immunoblotted for Enigma constructs with anti-FLAG and for Cbl-c as indicated on the right. GST pull-downs were immunoblotted for GST-Cbl-c with anti-GST and for each of the FLAG-Enigma proteins as indicated. Arrow indicates expected position of ΔLIM1-3 protein. Molecular weight standards are indicated on the left of each panel and Ponceau S staining serves as a measure of protein loading.

Techniques Used: Transfection, Plasmid Preparation, Molecular Weight, Staining, Immunoprecipitation, Construct

Cbl binds RET and MEN2A kinases. ( A ) HEK293T cells were transfected separately with pJ7Ω plasmids encoding each of the following four RET kinase isoforms: RET 9, RET 51, and their MEN2A (C634R) mutant isoforms. Each was transfected both with and without GST-Cbl-c. Whole cell lysates were collected at 48h post-transfection and GST pull-downs were performed on 300 µg of whole cell lysates and immunoblotted (IB) as indicated. Whole cell lysates were immunoblotted as indicated and molecular weight standards are indicated on the left of each panel β-actin serves as a loading control. ( B ) HEK293T cells were transfected with GST-Cbl-c both with and without RET9MEN2A. Replicate plates were treated with either 500 µM Sorafenib (+) or DMSO as vehicle control (-) for 90m prior to collection. Whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysates using anti-RET antibody with Protein A/G Sepharose and immunoblotted for RET and phosphotyrosine (pY) as indicated. GST pull-downs were performed, as described above, and immunoblotted (IB) for RET, phosphotyrosine (pY), and GST-Cbl-c as indicated. Molecular weight standards are indicated to the left of each panel and Hsc70 serve as loading controls.
Figure Legend Snippet: Cbl binds RET and MEN2A kinases. ( A ) HEK293T cells were transfected separately with pJ7Ω plasmids encoding each of the following four RET kinase isoforms: RET 9, RET 51, and their MEN2A (C634R) mutant isoforms. Each was transfected both with and without GST-Cbl-c. Whole cell lysates were collected at 48h post-transfection and GST pull-downs were performed on 300 µg of whole cell lysates and immunoblotted (IB) as indicated. Whole cell lysates were immunoblotted as indicated and molecular weight standards are indicated on the left of each panel β-actin serves as a loading control. ( B ) HEK293T cells were transfected with GST-Cbl-c both with and without RET9MEN2A. Replicate plates were treated with either 500 µM Sorafenib (+) or DMSO as vehicle control (-) for 90m prior to collection. Whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysates using anti-RET antibody with Protein A/G Sepharose and immunoblotted for RET and phosphotyrosine (pY) as indicated. GST pull-downs were performed, as described above, and immunoblotted (IB) for RET, phosphotyrosine (pY), and GST-Cbl-c as indicated. Molecular weight standards are indicated to the left of each panel and Hsc70 serve as loading controls.

Techniques Used: Transfection, Mutagenesis, Molecular Weight

37) Product Images from "Blue light and CO2 signals converge to regulate light-induced stomatal opening"

Article Title: Blue light and CO2 signals converge to regulate light-induced stomatal opening

Journal: Nature Communications

doi: 10.1038/s41467-017-01237-5

CBC1 and CBC2 are phosphorylated by HT1 kinase. a BiFC assays of CBCs and HT1. CBCs-nYFP and HT1-cYFP were co-transfected to MCPs. White bars represent 20 μm. b Pull-down assays of HT1 by CBC1 and CBC2. FLAG-CBCs and His-HT1 proteins were synthesized in vitro transcription/translation. c Autophosphorylation of GST-CBC1 and GST-CBC2. CBC1 (D271N) and CBC2 (D245N) contain the kinase-dead mutation. d In vitro kinase assay of CBC1 and CBC2. Artificial substrates of casein, MBP, and histone of 5 μg were used. e In vitro kinase assay of HT1, CBC1, and CBC2. Kinase-dead proteins of HT1 (D184N), CBC1 (D271N), and CBC2 (D245N) were used as substrates. One microgram of active kinase and substrate was used. f In vitro phosphorylation of CBC1 (D271N) and Ser-substituted CBC1 (D271N S43A S45A) by HT1. All GST-tagged proteins used in c – f were expressed in E. coli and purified with glutathione-Sepharose beads. Phosphorylation assays were done as in Fig. 4e
Figure Legend Snippet: CBC1 and CBC2 are phosphorylated by HT1 kinase. a BiFC assays of CBCs and HT1. CBCs-nYFP and HT1-cYFP were co-transfected to MCPs. White bars represent 20 μm. b Pull-down assays of HT1 by CBC1 and CBC2. FLAG-CBCs and His-HT1 proteins were synthesized in vitro transcription/translation. c Autophosphorylation of GST-CBC1 and GST-CBC2. CBC1 (D271N) and CBC2 (D245N) contain the kinase-dead mutation. d In vitro kinase assay of CBC1 and CBC2. Artificial substrates of casein, MBP, and histone of 5 μg were used. e In vitro kinase assay of HT1, CBC1, and CBC2. Kinase-dead proteins of HT1 (D184N), CBC1 (D271N), and CBC2 (D245N) were used as substrates. One microgram of active kinase and substrate was used. f In vitro phosphorylation of CBC1 (D271N) and Ser-substituted CBC1 (D271N S43A S45A) by HT1. All GST-tagged proteins used in c – f were expressed in E. coli and purified with glutathione-Sepharose beads. Phosphorylation assays were done as in Fig. 4e

Techniques Used: Bimolecular Fluorescence Complementation Assay, Transfection, Synthesized, In Vitro, Mutagenesis, Kinase Assay, Purification

Phosphorylation of CBC1 by phototropin1 in a BL-dependent manner. a BL-dependent mobility shift of CBC1. GCPs were illuminated with light as described in Fig. 1a . Samples were obtained 1 min after the start of the BL pulse. Immunoblot analysis of CBC1 was performed after electrophoresis on Phos-tag SDS-PAGE that contained 10% acrylamide, 20 μM Phos-tag, and 40 μM MnCl 2 . For CBC2, 40 μM Phos-tag and 80 μM MnCl 2 were included in the gel. b Phototropin-mediated phosphorylation of CBC1. CBC1 detection was done as described in a . c Pull-down assays of CBC1 and CBC2 by phot1. FLAG-phot1 and His-CBCs were synthesized in vitro transcription/translation. d BiFC assays of CBCs and phot1. CBCs-nYFP and phot1-cYFP were co-transfected to MCPs. White bars represent 20 μm. e Phosphorylation of kinase-dead CBC1 (D271N) and CBC2 (D245N) by P1C using [γ- 32 P] ATP. Phosphorylation assays were performed in 30 μl reaction mixtures that contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 3.3 μM ATP, 20 μCi of [γ- 32 P] ATP, and purified proteins. The reactions proceeded for 2 h at 15 °C. GST-tagged P1C or kinase-dead P1C (D806N) were included at 2.5 μg, and CBC1 (D271N) and CBC2 (D245N) were added at 1.2 μg. These GST proteins were expressed in E. coli and purified with glutathione-Sepharose beads. f Phosphorylation of kinase-dead CBC1 (D271N) and CBC1 (D271N S43A S45A) by P1C. Measurement was done as same as e . g Mobility of CBC1-GFP and CBC1 (S43A S45A)-GFP expressed in guard cells was determined as a , except the acrylamide concentration was used at 6%. The cbc1 cbc2 double mutant was transformed with CBC-GFP or CBC1 ( S43A S45A ) -GFP and GCPs were prepared from these transgenic plants. h Pull-down assays of His-CBC1 and His-CBC2 by FLAG-tagged CBC1 and CBC2. Tagged proteins were synthesized by in vitro transcription/translation. i BL-dependent phosphorylation of CBC1 in the blus1-3 mutant. CBC1 detection was done as described in a
Figure Legend Snippet: Phosphorylation of CBC1 by phototropin1 in a BL-dependent manner. a BL-dependent mobility shift of CBC1. GCPs were illuminated with light as described in Fig. 1a . Samples were obtained 1 min after the start of the BL pulse. Immunoblot analysis of CBC1 was performed after electrophoresis on Phos-tag SDS-PAGE that contained 10% acrylamide, 20 μM Phos-tag, and 40 μM MnCl 2 . For CBC2, 40 μM Phos-tag and 80 μM MnCl 2 were included in the gel. b Phototropin-mediated phosphorylation of CBC1. CBC1 detection was done as described in a . c Pull-down assays of CBC1 and CBC2 by phot1. FLAG-phot1 and His-CBCs were synthesized in vitro transcription/translation. d BiFC assays of CBCs and phot1. CBCs-nYFP and phot1-cYFP were co-transfected to MCPs. White bars represent 20 μm. e Phosphorylation of kinase-dead CBC1 (D271N) and CBC2 (D245N) by P1C using [γ- 32 P] ATP. Phosphorylation assays were performed in 30 μl reaction mixtures that contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 3.3 μM ATP, 20 μCi of [γ- 32 P] ATP, and purified proteins. The reactions proceeded for 2 h at 15 °C. GST-tagged P1C or kinase-dead P1C (D806N) were included at 2.5 μg, and CBC1 (D271N) and CBC2 (D245N) were added at 1.2 μg. These GST proteins were expressed in E. coli and purified with glutathione-Sepharose beads. f Phosphorylation of kinase-dead CBC1 (D271N) and CBC1 (D271N S43A S45A) by P1C. Measurement was done as same as e . g Mobility of CBC1-GFP and CBC1 (S43A S45A)-GFP expressed in guard cells was determined as a , except the acrylamide concentration was used at 6%. The cbc1 cbc2 double mutant was transformed with CBC-GFP or CBC1 ( S43A S45A ) -GFP and GCPs were prepared from these transgenic plants. h Pull-down assays of His-CBC1 and His-CBC2 by FLAG-tagged CBC1 and CBC2. Tagged proteins were synthesized by in vitro transcription/translation. i BL-dependent phosphorylation of CBC1 in the blus1-3 mutant. CBC1 detection was done as described in a

Techniques Used: Mobility Shift, Electrophoresis, SDS Page, Synthesized, In Vitro, Bimolecular Fluorescence Complementation Assay, Transfection, Purification, Concentration Assay, Mutagenesis, Transformation Assay, Transgenic Assay

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0161672

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

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

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

Techniques Used: Western Blot, Recombinant, Binding Assay

39) Product Images from "Cellular and viral peptides bind multiple sites on the N-terminal domain of clathrin"

Article Title: Cellular and viral peptides bind multiple sites on the N-terminal domain of clathrin

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/tra.12457

; AmphCBM, human amphiphysin I CBM; AP2CBM, CBM from flexible hinge of β2 adaptin subunit of human AP2; HDAg‐L1, putative CBM from clade I hepatitis D virus large antigen; HDAg‐L2, putative CBM from clade II hepatitis D virus large antigen; Wbox, human amphiphysin W box binding motif. B, Capture (“GST pull‐down”) of purified clathrin by GST‐tagged clathrin‐binding peptides. Clathrin (input) was incubated with glutathione sepharose pre‐loaded with GST‐tagged “bait” proteins. After washing, proteins bound to the beads (pellet) were subjected to SDS‐PAGE and immunoblotting (WB) using an antibody that recognizes clathrin NTD (αNTD). C, Capture of His‐NTD‐NEMO by GST‐tagged clathrin binding peptides. Purified recombinant His‐NTD‐NEMO was used in GST pull‐down experiments as in (B).
Figure Legend Snippet: ; AmphCBM, human amphiphysin I CBM; AP2CBM, CBM from flexible hinge of β2 adaptin subunit of human AP2; HDAg‐L1, putative CBM from clade I hepatitis D virus large antigen; HDAg‐L2, putative CBM from clade II hepatitis D virus large antigen; Wbox, human amphiphysin W box binding motif. B, Capture (“GST pull‐down”) of purified clathrin by GST‐tagged clathrin‐binding peptides. Clathrin (input) was incubated with glutathione sepharose pre‐loaded with GST‐tagged “bait” proteins. After washing, proteins bound to the beads (pellet) were subjected to SDS‐PAGE and immunoblotting (WB) using an antibody that recognizes clathrin NTD (αNTD). C, Capture of His‐NTD‐NEMO by GST‐tagged clathrin binding peptides. Purified recombinant His‐NTD‐NEMO was used in GST pull‐down experiments as in (B).

Techniques Used: Binding Assay, Purification, Incubation, SDS Page, Western Blot, Recombinant

The overlapping β2 adaptin arrestin‐box and clathrin‐box motifs both bind multiple sites on clathrin N‐terminal domain (NTD). A, Glutathione S‐transferase (GST) fusions of the clathrin‐box motif (GST‐AP2CBM) and arrestin‐binding motif (GST‐AP2arrS and GST‐AP2arrL) from the hinge region of β2 adaptin, the arrestin‐box motif constructs having either the next residue of β2 adaptin (“L”, GST‐AP2arrS) or the sequence that follows the LLGDL motif of arrestin2L (“ASS”, GST‐AP2arrL) appended at their C termini. B, Capture of wild‐type NTD or a mutant with disrupted clathrin and Royle boxes (Q89A + F91K + F9W) by GST‐tagged β2 adaptin clathrin‐binding motifs. Purified recombinant wild‐type or mutant His‐NTD‐NEMO was incubated with glutathione sepharose pre‐loaded with GST‐tagged “bait” proteins. After washing, proteins bound to the beads (pellet) were subjected to SDS‐PAGE and immunoblotting (WB) using an antibody that recognizes clathrin NTD (αNTD).
Figure Legend Snippet: The overlapping β2 adaptin arrestin‐box and clathrin‐box motifs both bind multiple sites on clathrin N‐terminal domain (NTD). A, Glutathione S‐transferase (GST) fusions of the clathrin‐box motif (GST‐AP2CBM) and arrestin‐binding motif (GST‐AP2arrS and GST‐AP2arrL) from the hinge region of β2 adaptin, the arrestin‐box motif constructs having either the next residue of β2 adaptin (“L”, GST‐AP2arrS) or the sequence that follows the LLGDL motif of arrestin2L (“ASS”, GST‐AP2arrL) appended at their C termini. B, Capture of wild‐type NTD or a mutant with disrupted clathrin and Royle boxes (Q89A + F91K + F9W) by GST‐tagged β2 adaptin clathrin‐binding motifs. Purified recombinant wild‐type or mutant His‐NTD‐NEMO was incubated with glutathione sepharose pre‐loaded with GST‐tagged “bait” proteins. After washing, proteins bound to the beads (pellet) were subjected to SDS‐PAGE and immunoblotting (WB) using an antibody that recognizes clathrin NTD (αNTD).

Techniques Used: Binding Assay, Construct, Sequencing, Mutagenesis, Purification, Recombinant, Incubation, SDS Page, Western Blot

40) Product Images from "Tetraspanin-decorated extracellular vesicle-mimetics as a novel adaptable reference material"

Article Title: Tetraspanin-decorated extracellular vesicle-mimetics as a novel adaptable reference material

Journal: Journal of Extracellular Vesicles

doi: 10.1080/20013078.2019.1573052

Production of recombinant tetraspanin-LELs. (a) Schematic illustration of the affinity chromatography method used for recombinant tetraspanin LELs purification using glutathione-sepharose beads, thrombin and benzamidine-sepharose beads. (b) Follow up of recombinant tetraspanin LELs production. The purification process was followed by Coomassie Blue staining after SDS-PAGE separation. Lanes 1–3: Samples of 10 µL Glutathione-sepharose beads before Thrombin treatment. Lanes 5–7: 10 µL of Glutathione-sepharose beads after treatment with Thrombin. Lanes 8–10: 20 µL of each supernatant after benzamidine-sepharose removal of Thrombin. Red arrows indicate the final LEL biotinylated product. (c) Western blot analysis of recombinant tetraspanin LELs. Samples were subjected to SDS-PAGE and detected by chemiluminiscence using Avidin-Biotin-HRP Complexes. Lanes 2–4: Samples of 10 µL of AviLELAvi-GST of CD9, CD63 and CD81 coupled to Glutathione-sepharose. Lanes 5–7: Samples of 20 µL of AviCD9LELAvi, AviCD63LELAvi and AviCD81LELAvi. Lanes 8–10 Samples of 10 µL of Glutathione-sepharose beads after Thrombin treatment. Red arrows indicate the final LEL biotinylated product. (d) Dot blot immunodetection of recombinant tetraspanin LELs. The final products, biotinylated CD9, CD63 and CD81 LELs, were detected by Dot-blot using ABC Peroxidase Standard Staining Kit and monoclonal antibodies directed against CD9 (clone VJ 1/20), CD63 (TEA 3/10) and CD81 (5A6).
Figure Legend Snippet: Production of recombinant tetraspanin-LELs. (a) Schematic illustration of the affinity chromatography method used for recombinant tetraspanin LELs purification using glutathione-sepharose beads, thrombin and benzamidine-sepharose beads. (b) Follow up of recombinant tetraspanin LELs production. The purification process was followed by Coomassie Blue staining after SDS-PAGE separation. Lanes 1–3: Samples of 10 µL Glutathione-sepharose beads before Thrombin treatment. Lanes 5–7: 10 µL of Glutathione-sepharose beads after treatment with Thrombin. Lanes 8–10: 20 µL of each supernatant after benzamidine-sepharose removal of Thrombin. Red arrows indicate the final LEL biotinylated product. (c) Western blot analysis of recombinant tetraspanin LELs. Samples were subjected to SDS-PAGE and detected by chemiluminiscence using Avidin-Biotin-HRP Complexes. Lanes 2–4: Samples of 10 µL of AviLELAvi-GST of CD9, CD63 and CD81 coupled to Glutathione-sepharose. Lanes 5–7: Samples of 20 µL of AviCD9LELAvi, AviCD63LELAvi and AviCD81LELAvi. Lanes 8–10 Samples of 10 µL of Glutathione-sepharose beads after Thrombin treatment. Red arrows indicate the final LEL biotinylated product. (d) Dot blot immunodetection of recombinant tetraspanin LELs. The final products, biotinylated CD9, CD63 and CD81 LELs, were detected by Dot-blot using ABC Peroxidase Standard Staining Kit and monoclonal antibodies directed against CD9 (clone VJ 1/20), CD63 (TEA 3/10) and CD81 (5A6).

Techniques Used: Recombinant, Affinity Chromatography, Purification, Staining, SDS Page, Western Blot, Avidin-Biotin Assay, Dot Blot, Immunodetection

Optimisation of the purification process of the recombinant biotinylated tetraspanin-LEL peptides. (a) Cell lysis optimisation. Six different lysis conditions (indicated in Table 2 ) were tested. 10 µL of the obtained AviCD63LELAvi-GST coupled to Glutathione-sepharose beads were lysed in Laemmli buffer, subjected to SDS-PAGE and analysed by Coomassie Blue staining. (b) Efficiency of recombinant protein recovery. Non-soluble fraction of the bacterial lysates obtained with lysis condition 2, as well as the recombinant affinity purified AviLELAvi-GST proteins, were tested by SDS-PAGE to check the recovery of GST fusion proteins from E.coli . Lanes 1–3: Bacterial lysates. Lanes 8–10: 10 µL of LELs-GST-Glutathione-sepharose. (c) Final optimisation of lysis conditions. Total bacterial lysates obtained using condition 7, as well as the recombinant affinity purified AviLELAvi-GST proteins were analysed by SDS-PAGE. Lanes 1,2,4: Non-soluble bacterial lysates. Lanes 7–9: 10 µL of LELs-GST-Glutathione-sepharose obtained. Red arrows indicate the expected molecular weight for the recombinant fusion tetraspanins, which is 37 kDa. (d) Optimisation of D-biotin labelling. Dot Blot analysis using an ABC Peroxidase Standard Kit and anti-CD63 specific antibody (clone TEA 3/10 ) for detection of the indicated volumes of bacterial cell lysates from AviCD63LELAvi-GST-transformed cultures grown in the presence of the indicated biotin concentrations.
Figure Legend Snippet: Optimisation of the purification process of the recombinant biotinylated tetraspanin-LEL peptides. (a) Cell lysis optimisation. Six different lysis conditions (indicated in Table 2 ) were tested. 10 µL of the obtained AviCD63LELAvi-GST coupled to Glutathione-sepharose beads were lysed in Laemmli buffer, subjected to SDS-PAGE and analysed by Coomassie Blue staining. (b) Efficiency of recombinant protein recovery. Non-soluble fraction of the bacterial lysates obtained with lysis condition 2, as well as the recombinant affinity purified AviLELAvi-GST proteins, were tested by SDS-PAGE to check the recovery of GST fusion proteins from E.coli . Lanes 1–3: Bacterial lysates. Lanes 8–10: 10 µL of LELs-GST-Glutathione-sepharose. (c) Final optimisation of lysis conditions. Total bacterial lysates obtained using condition 7, as well as the recombinant affinity purified AviLELAvi-GST proteins were analysed by SDS-PAGE. Lanes 1,2,4: Non-soluble bacterial lysates. Lanes 7–9: 10 µL of LELs-GST-Glutathione-sepharose obtained. Red arrows indicate the expected molecular weight for the recombinant fusion tetraspanins, which is 37 kDa. (d) Optimisation of D-biotin labelling. Dot Blot analysis using an ABC Peroxidase Standard Kit and anti-CD63 specific antibody (clone TEA 3/10 ) for detection of the indicated volumes of bacterial cell lysates from AviCD63LELAvi-GST-transformed cultures grown in the presence of the indicated biotin concentrations.

Techniques Used: Purification, Recombinant, Lysis, SDS Page, Staining, Affinity Purification, Molecular Weight, Dot Blot, Transformation Assay

41) Product Images from "Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture"

Article Title: Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm553

Preparation of RNase MRP protein subunits. ( A ) Expressed GST-fusion proteins bound to glutathione Sepharose 4B beads. The expression and purity of GST-fusion protein preparations were determined by SDS-PAGE analysis and Coomassie staining. The asterisks (*) indicate the full-length GST-(fusion) proteins. The bands seen beneath the full-length GST-(fusion) proteins probably represent truncated versions of the full-length recombinant proteins. The sizes of the molecular weight markers are shown on the right. ( B ) Radiolabelled, cleaved proteins. Whilst bound to glutathione Sepharose, GST fusions were treated with bovine heart kinase in the presence of γ- 32 P-ATP to achieve radiolabelling, followed by removal of the GST-tag by overnight cleavage with PreScission protease. The efficiency of radiolabelling and the purity of the cleaved proteins were assessed by SDS-PAGE analysis followed by exposure to PhosphoImager screens and analysis using a Typhoon scanner. The asterisks (*) indicate the radiolabelled, untagged proteins. The sizes of the molecular weight markers are shown on the left.
Figure Legend Snippet: Preparation of RNase MRP protein subunits. ( A ) Expressed GST-fusion proteins bound to glutathione Sepharose 4B beads. The expression and purity of GST-fusion protein preparations were determined by SDS-PAGE analysis and Coomassie staining. The asterisks (*) indicate the full-length GST-(fusion) proteins. The bands seen beneath the full-length GST-(fusion) proteins probably represent truncated versions of the full-length recombinant proteins. The sizes of the molecular weight markers are shown on the right. ( B ) Radiolabelled, cleaved proteins. Whilst bound to glutathione Sepharose, GST fusions were treated with bovine heart kinase in the presence of γ- 32 P-ATP to achieve radiolabelling, followed by removal of the GST-tag by overnight cleavage with PreScission protease. The efficiency of radiolabelling and the purity of the cleaved proteins were assessed by SDS-PAGE analysis followed by exposure to PhosphoImager screens and analysis using a Typhoon scanner. The asterisks (*) indicate the radiolabelled, untagged proteins. The sizes of the molecular weight markers are shown on the left.

Techniques Used: Expressing, SDS Page, Staining, Recombinant, Molecular Weight

Protein–protein interactions. The figure shows a typical set of interaction data for two protein subunits. The protein subunit in untagged radiolabelled form was screened for interaction with each RNase MRP GST-fusion protein immobilized on glutathione Sepharose beads in a phosphate buffer containing 150 mM NaCl. GST was included as a control. The first lane in each case contains untagged radiolabelled protein only, indicating the total (100%) amount of input. ( A ) Interactions of Pop1p, ( B ) interactions of Rmp1p. Protein–protein interactions were quantitated using a Typhoon scanner and ImageQuant software. Interactions were assigned as strong (‘+’; at least 40% of input protein co-precipitated), weak (‘+’/−; between 20 and 40% of input protein co-precipitated) or none (‘−’;
Figure Legend Snippet: Protein–protein interactions. The figure shows a typical set of interaction data for two protein subunits. The protein subunit in untagged radiolabelled form was screened for interaction with each RNase MRP GST-fusion protein immobilized on glutathione Sepharose beads in a phosphate buffer containing 150 mM NaCl. GST was included as a control. The first lane in each case contains untagged radiolabelled protein only, indicating the total (100%) amount of input. ( A ) Interactions of Pop1p, ( B ) interactions of Rmp1p. Protein–protein interactions were quantitated using a Typhoon scanner and ImageQuant software. Interactions were assigned as strong (‘+’; at least 40% of input protein co-precipitated), weak (‘+’/−; between 20 and 40% of input protein co-precipitated) or none (‘−’;

Techniques Used: Software

42) Product Images from "UFL1 promotes histone H4 ufmylation and ATM activation"

Article Title: UFL1 promotes histone H4 ufmylation and ATM activation

Journal: Nature Communications

doi: 10.1038/s41467-019-09175-0

ATM phosphorylates UFL1 and enhances its activity. a Flag-UFL1 was expressed in U2OS cells. Following vehicle or ATM inhibitor KU55933 treatment, the cells were irradiated with IR. Flag-UFL1 was immunoprecipitated and blots were probed with phospho-SQ/TQ antibody and Flag antibody. b Flag-UFL1 was expressed in atm + / + or atm−/− MEF cells. Cells were irradiated with IR and UFL1 phosphorylation was then examined as in a . c Flag-UFL1 or the S462A mutant was expressed in U2OS cells. Cells were treated with or without 10 Gy IR. Thirty minutes later, cells were lysed and incubated with Flag antibody-conjugated agarose beads. The immunoprecipitates were blotted with phospho-SQ/TQ antibody and Flag antibody. d Flag-tagged wildtype (WT) and S462A mutant UFL1 expressing cells were treated with or without 2 Gy IR and fixed and stained with indicated antibodies. Scale bars, 10 µm. e Flag-tagged UFL1 or S462A mutant expressing U2OS cells were irradiated with 10 Gy IR. Thirty minutes later, UFL1 WT and the S462A mutant were purified from the cells, and incubated with purified UBA5, UFC1, UFL1, UFM1, and H4 protein at 30 ° C for 90 min. The reaction was assessed by probing the blot with the indicated antibody. f His-UFM1 was expressed in ATM proficient or deficient cells. His-UFM1 conjugated protein was purified under denaturing conditions. The purified samples were blotted with indicated antibodies. g Flag-tagged UFL1 or the S462A mutant was expressed in U2OS cells. Cells were harvested at indicated time points following IR and blotted with indicated antibodies. h Colony formation assay following IR was performed with UFL1-depleted cells reconstituted with WT UFL1 or the S462A mutant. The data is presented as mean±s.e.m. of n = 3 independent experiments. Dots depict individual data points. Statistical significance was calculated using two-way ANOVA. ** P
Figure Legend Snippet: ATM phosphorylates UFL1 and enhances its activity. a Flag-UFL1 was expressed in U2OS cells. Following vehicle or ATM inhibitor KU55933 treatment, the cells were irradiated with IR. Flag-UFL1 was immunoprecipitated and blots were probed with phospho-SQ/TQ antibody and Flag antibody. b Flag-UFL1 was expressed in atm + / + or atm−/− MEF cells. Cells were irradiated with IR and UFL1 phosphorylation was then examined as in a . c Flag-UFL1 or the S462A mutant was expressed in U2OS cells. Cells were treated with or without 10 Gy IR. Thirty minutes later, cells were lysed and incubated with Flag antibody-conjugated agarose beads. The immunoprecipitates were blotted with phospho-SQ/TQ antibody and Flag antibody. d Flag-tagged wildtype (WT) and S462A mutant UFL1 expressing cells were treated with or without 2 Gy IR and fixed and stained with indicated antibodies. Scale bars, 10 µm. e Flag-tagged UFL1 or S462A mutant expressing U2OS cells were irradiated with 10 Gy IR. Thirty minutes later, UFL1 WT and the S462A mutant were purified from the cells, and incubated with purified UBA5, UFC1, UFL1, UFM1, and H4 protein at 30 ° C for 90 min. The reaction was assessed by probing the blot with the indicated antibody. f His-UFM1 was expressed in ATM proficient or deficient cells. His-UFM1 conjugated protein was purified under denaturing conditions. The purified samples were blotted with indicated antibodies. g Flag-tagged UFL1 or the S462A mutant was expressed in U2OS cells. Cells were harvested at indicated time points following IR and blotted with indicated antibodies. h Colony formation assay following IR was performed with UFL1-depleted cells reconstituted with WT UFL1 or the S462A mutant. The data is presented as mean±s.e.m. of n = 3 independent experiments. Dots depict individual data points. Statistical significance was calculated using two-way ANOVA. ** P

Techniques Used: Activity Assay, Irradiation, Immunoprecipitation, Mutagenesis, Incubation, Expressing, Staining, Purification, Colony Assay

UFL1 monoufmylates histone H4 and promotes ATM activation. a Selected proteins identified by mass spectrometry from irradiated 293T cell expressing Flag-His vector or Flag-His-UFM1. N = 1 sample in each group was analyzed. The full list of identified proteins is provided in Supplementary Data 1 . Among histone proteins, only H4 is enriched in the Flag-His-UFM1 purification (9 unique/20 total peptides) compared to the Flag-His purification (6 unique/7 total peptides), suggesting that H4 might be ufmylated. b Flag-His-ufmylated proteins were purified from 293T cells before and after IR (2 Gy) after purification with nickel beads and anti-Flag agarose. The immunoprecipitates were detected with indicated antibodies. c Flag-His-ufmylated H4 was purified from control and UFL1 knockdown cells with or without 2 Gy IR and blotted with indicated antibodies. d In vitro ufmylation assay. Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl 2 at 30 ° C for 90 min. The reaction products were probed with indicated antibodies. e Wildtype (WT) histone H4 and 11 different single lysine (K) to arginine (R) mutants were transfected into U2OS cells. Flag and His tandem purification was performed and H4 ufmylation was analyzed. f Constructs expressing WT or K31R H4 were transfected into U2OS tet-on UFL1 shRNA expressing cells, and the cells were treated with doxycycline as indicated. Thirty minutes after 2 Gy IR, the cells were harvested and blotted with indicated antibodies. g Colony formation of U2OS cells expressing WT H4 or H4K31R following IR. The data presented are mean ± s.e.m. for n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA. * p
Figure Legend Snippet: UFL1 monoufmylates histone H4 and promotes ATM activation. a Selected proteins identified by mass spectrometry from irradiated 293T cell expressing Flag-His vector or Flag-His-UFM1. N = 1 sample in each group was analyzed. The full list of identified proteins is provided in Supplementary Data 1 . Among histone proteins, only H4 is enriched in the Flag-His-UFM1 purification (9 unique/20 total peptides) compared to the Flag-His purification (6 unique/7 total peptides), suggesting that H4 might be ufmylated. b Flag-His-ufmylated proteins were purified from 293T cells before and after IR (2 Gy) after purification with nickel beads and anti-Flag agarose. The immunoprecipitates were detected with indicated antibodies. c Flag-His-ufmylated H4 was purified from control and UFL1 knockdown cells with or without 2 Gy IR and blotted with indicated antibodies. d In vitro ufmylation assay. Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl 2 at 30 ° C for 90 min. The reaction products were probed with indicated antibodies. e Wildtype (WT) histone H4 and 11 different single lysine (K) to arginine (R) mutants were transfected into U2OS cells. Flag and His tandem purification was performed and H4 ufmylation was analyzed. f Constructs expressing WT or K31R H4 were transfected into U2OS tet-on UFL1 shRNA expressing cells, and the cells were treated with doxycycline as indicated. Thirty minutes after 2 Gy IR, the cells were harvested and blotted with indicated antibodies. g Colony formation of U2OS cells expressing WT H4 or H4K31R following IR. The data presented are mean ± s.e.m. for n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA. * p

Techniques Used: Activation Assay, Mass Spectrometry, Irradiation, Expressing, Plasmid Preparation, Purification, In Vitro, Incubation, Transfection, Construct, shRNA

43) Product Images from "Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments"

Article Title: Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments

Journal: Molecular Cell

doi: 10.1016/j.molcel.2017.07.001

RVFs and Nascent DNA Degradation (A) Residual biotin-dUTP in nuclei replicated in extracts treated as shown. Where indicated, extracts were Smarcal1-depleted and supplemented with buffer or 5 ng/μL recombinant Smarcal1 WT or Smarcal1 HD proteins. The fluorescence intensity of mock at 0 min was considered as 100%. (B) ELISA detection of BrdU in nascent ssDNA in nuclei incubated in extracts treated as shown. Mean intensity values ± SD (n = 3) are shown. (C and D) Gel showing the effect of Rad51 WT and Rad51 T131P (C) or RPA (D) pre-incubation with 5′ fluorescently labeled RVF, shown in the scheme, and subsequent incubation with Mre11. Reactions were resolved on denaturing 30% polyacrylamide gel. (E) Mre11-dependent DNA degradation rates in the presence of Rad51 WT , Rad51 T131P , or RPA relative to the amount of substrate (20 nM) shown in (C), lane (−), which was considered as 100%. Mean values ± SD (n = 3) are shown. (F) DNA binding of Rad51 WT or RPA to the same fluorescently labeled RVF DNA substrate (20 nM) resolved on 0.8% agarose gel. See also Figure S6 .
Figure Legend Snippet: RVFs and Nascent DNA Degradation (A) Residual biotin-dUTP in nuclei replicated in extracts treated as shown. Where indicated, extracts were Smarcal1-depleted and supplemented with buffer or 5 ng/μL recombinant Smarcal1 WT or Smarcal1 HD proteins. The fluorescence intensity of mock at 0 min was considered as 100%. (B) ELISA detection of BrdU in nascent ssDNA in nuclei incubated in extracts treated as shown. Mean intensity values ± SD (n = 3) are shown. (C and D) Gel showing the effect of Rad51 WT and Rad51 T131P (C) or RPA (D) pre-incubation with 5′ fluorescently labeled RVF, shown in the scheme, and subsequent incubation with Mre11. Reactions were resolved on denaturing 30% polyacrylamide gel. (E) Mre11-dependent DNA degradation rates in the presence of Rad51 WT , Rad51 T131P , or RPA relative to the amount of substrate (20 nM) shown in (C), lane (−), which was considered as 100%. Mean values ± SD (n = 3) are shown. (F) DNA binding of Rad51 WT or RPA to the same fluorescently labeled RVF DNA substrate (20 nM) resolved on 0.8% agarose gel. See also Figure S6 .

Techniques Used: Recombinant, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation, Recombinase Polymerase Amplification, Labeling, Binding Assay, Agarose Gel Electrophoresis

Brca2- and Rad51-Mediated Protection from Mre11 (A) Top: experimental scheme. Bottom: relative percentage of residual biotin-dUTP in sperm nuclei quantified using a fluorescence method. The fluorescence intensity of mock at 0 min was considered as 100%. Extracts were treated as indicated and supplemented with 100 μM mirin or recombinant Brca2c or Brca2d. Mean values ± SD (n = 3) are shown. (B and C) Gel showing the effect of Rad51 WT and Rad51 T131P (B) or RPA complex (C) pre-incubation with 5′ fluorescently labeled DNA substrate (20 nM), shown in the scheme containing phosphorothioate bonds (s), and subsequent incubation with Mre11 (30 nM). Reactions were resolved on denaturing 30% polyacrylamide gel. (D) Mre11-dependent DNA degradation rates in the presence of Rad51 WT , Rad51 T131P , or RPA relative to the amount of substrate shown in (B), lane (−), which was considered as 100%. Mean values ± SD (n = 3) are shown. (E) Electrophoretic mobility shift assay showing binding of Rad51 WT , Rad51 T131P , or RPA to the same fluorescently labeled DNA substrate (20 nM) resolved on 0.8% agarose gel. See also Figures S4 and S5 .
Figure Legend Snippet: Brca2- and Rad51-Mediated Protection from Mre11 (A) Top: experimental scheme. Bottom: relative percentage of residual biotin-dUTP in sperm nuclei quantified using a fluorescence method. The fluorescence intensity of mock at 0 min was considered as 100%. Extracts were treated as indicated and supplemented with 100 μM mirin or recombinant Brca2c or Brca2d. Mean values ± SD (n = 3) are shown. (B and C) Gel showing the effect of Rad51 WT and Rad51 T131P (B) or RPA complex (C) pre-incubation with 5′ fluorescently labeled DNA substrate (20 nM), shown in the scheme containing phosphorothioate bonds (s), and subsequent incubation with Mre11 (30 nM). Reactions were resolved on denaturing 30% polyacrylamide gel. (D) Mre11-dependent DNA degradation rates in the presence of Rad51 WT , Rad51 T131P , or RPA relative to the amount of substrate shown in (B), lane (−), which was considered as 100%. Mean values ± SD (n = 3) are shown. (E) Electrophoretic mobility shift assay showing binding of Rad51 WT , Rad51 T131P , or RPA to the same fluorescently labeled DNA substrate (20 nM) resolved on 0.8% agarose gel. See also Figures S4 and S5 .

Techniques Used: Fluorescence, Recombinant, Recombinase Polymerase Amplification, Incubation, Labeling, Electrophoretic Mobility Shift Assay, Binding Assay, Agarose Gel Electrophoresis

44) Product Images from "The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions"

Article Title: The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0177879

PET 52-233 of testin directly interacts with LIM1-3 domains in vitro . A-D) The experimental setup is similar as shown in Fig 2A . A recombinant GST-testin variant used as bait (GST-CR (A), GST-LIM1-3 (B, C) or GST-ΔPET (D)) was trapped on glutathione resin and presented in immobilised form to a second untagged testin-variant in solution used as prey (LIM1-3 (A, C), PET 52-233 (B) or ΔPET (D)). Coomassie stained SDS-PAGE analysis (A-C) or western blot analysis (D) using anti-testin (green) and anti-GST (red) antibodies is shown. Input (I) shows the untagged prey protein prior to incubation with the resin. Lanes indicated with P show the proteins present on the resin (immobilized bait and/or bound prey) after the incubation. GST resin (D) or GST-cofilin resin (A-C) incubated with the same untagged testin variant as prey were used as negative controls. Untagged protein bound to the GST-testin variant immobilised on the resin is highlighted by a red box (B). Positions of bait, prey and negative control (Neg Ctr) bait are indicated in each panel, M: marker proteins (kDa). E) The indicated concentrations of GST-LIM1-3 immobilized on glutathione-sepharose beads were prepared and incubated with 2.5μM PET 52-233 . For each LIM1-3 concentration (indicated as [GST-LIM1-3] total (μM)), the level of unbound PET 52-233 was analysed by Coomassie staining after SDS-PAGE (left). Aspecific binding was assessed by incubation of glutathione-sepharose beads, lacking LIM1-3, with a similar concentration of PET 52-233 I: input, representing 2.5 μM PET 52-233 in solution without incubation to LIM-1-3 coupled glutathione beads (reference for unbound 100% or bound 0%), A: aspecific binding of the PET 52-233 ligand to beads without LIM1-3, M: molecular weight marker (kDa). (right) The % amounts of bound PET 52-233 calculated from the measured intensities of the unbound material on gel (left) were plotted versus GST-LIM1-3 concentrations (graph right, red dots). The amount of aspecifically bound PET 52-233 is represented by a black dot. The solid blue line in the graph is the fitted curve taking into account aspecific binding (see Materials and methods for details).
Figure Legend Snippet: PET 52-233 of testin directly interacts with LIM1-3 domains in vitro . A-D) The experimental setup is similar as shown in Fig 2A . A recombinant GST-testin variant used as bait (GST-CR (A), GST-LIM1-3 (B, C) or GST-ΔPET (D)) was trapped on glutathione resin and presented in immobilised form to a second untagged testin-variant in solution used as prey (LIM1-3 (A, C), PET 52-233 (B) or ΔPET (D)). Coomassie stained SDS-PAGE analysis (A-C) or western blot analysis (D) using anti-testin (green) and anti-GST (red) antibodies is shown. Input (I) shows the untagged prey protein prior to incubation with the resin. Lanes indicated with P show the proteins present on the resin (immobilized bait and/or bound prey) after the incubation. GST resin (D) or GST-cofilin resin (A-C) incubated with the same untagged testin variant as prey were used as negative controls. Untagged protein bound to the GST-testin variant immobilised on the resin is highlighted by a red box (B). Positions of bait, prey and negative control (Neg Ctr) bait are indicated in each panel, M: marker proteins (kDa). E) The indicated concentrations of GST-LIM1-3 immobilized on glutathione-sepharose beads were prepared and incubated with 2.5μM PET 52-233 . For each LIM1-3 concentration (indicated as [GST-LIM1-3] total (μM)), the level of unbound PET 52-233 was analysed by Coomassie staining after SDS-PAGE (left). Aspecific binding was assessed by incubation of glutathione-sepharose beads, lacking LIM1-3, with a similar concentration of PET 52-233 I: input, representing 2.5 μM PET 52-233 in solution without incubation to LIM-1-3 coupled glutathione beads (reference for unbound 100% or bound 0%), A: aspecific binding of the PET 52-233 ligand to beads without LIM1-3, M: molecular weight marker (kDa). (right) The % amounts of bound PET 52-233 calculated from the measured intensities of the unbound material on gel (left) were plotted versus GST-LIM1-3 concentrations (graph right, red dots). The amount of aspecifically bound PET 52-233 is represented by a black dot. The solid blue line in the graph is the fitted curve taking into account aspecific binding (see Materials and methods for details).

Techniques Used: Positron Emission Tomography, In Vitro, Recombinant, Variant Assay, Staining, SDS Page, Western Blot, Incubation, Negative Control, Marker, Concentration Assay, Binding Assay, Molecular Weight

Full length testin interacts with full length testin in vitro. A) General Scheme of affinity purification used to produce data in several figures, to demonstrate an interaction of testin with other testin variants. In panel B of this figure, recombinant GST-FL was either used as bait on glutathione-sepharose or in parallel treated with thrombin to remove GST to be used as prey in an untagged form. Thrombin was inactivated prior to addition of this soluble form (input: I) to the resin with GST bound protein. After washing the resin, bound proteins were eluted with heated sample buffer and thus contain both bait and potential prey proteins (affinity purified: P). Proteins were detected either by Western Blotting (Figs 2B–2F and 4D ) or Coomassie (Figs 4A–4C and 5A–5D ), T = temperature. B) Immobilised recombinant GST-FL (bait) was incubated with soluble recombinant untagged FL (prey). A Western blot using anti-testin (green) and anti-GST (red) antibodies of the input (I) and proteins on the resin (P) is shown. FL prey (approx. 50 kDa) was present on the resin together with the bait GST-FL (lane ‘GST-FL/P’). Input (I) shows the untagged FL in solution. Recombinant immobilised GST was used as a negative control (lane ‘GST/P’). C) Western blot analysis (anti-testin (green), anti-GST (red)) of a mock buffer control incubated with immobilised GST-FL on resin. Similar as in B, the buffer contains inactivated thrombin but no soluble FL prey. Untagged FL is absent on the resin (P) (compare to Fig 2B , lane ‘GST-FL/P’) indicating that possible residual thrombin activity is not cleaving the GST-FL on the resin. D, E, F) Immobilised recombinant GST-FL (bait) was incubated with soluble untagged Evl (positive control, D) or cofilin (negative control, F) as preys. Immobilised GST was incubated with Evl (prey) and used as additional negative control (E). Western blot analysis of inputs (I) and proteins on the resin (P) is shown using anti testin (green), anti-Evl, anti-cofilin and anti-GST (red) antibodies. Untagged Evl (prey) is present on the GST-FL resin (lane P, 2D). Positions of bait, prey and negative control bait (Neg Ctr) are indicated in each panel. M: marker proteins (kDa).
Figure Legend Snippet: Full length testin interacts with full length testin in vitro. A) General Scheme of affinity purification used to produce data in several figures, to demonstrate an interaction of testin with other testin variants. In panel B of this figure, recombinant GST-FL was either used as bait on glutathione-sepharose or in parallel treated with thrombin to remove GST to be used as prey in an untagged form. Thrombin was inactivated prior to addition of this soluble form (input: I) to the resin with GST bound protein. After washing the resin, bound proteins were eluted with heated sample buffer and thus contain both bait and potential prey proteins (affinity purified: P). Proteins were detected either by Western Blotting (Figs 2B–2F and 4D ) or Coomassie (Figs 4A–4C and 5A–5D ), T = temperature. B) Immobilised recombinant GST-FL (bait) was incubated with soluble recombinant untagged FL (prey). A Western blot using anti-testin (green) and anti-GST (red) antibodies of the input (I) and proteins on the resin (P) is shown. FL prey (approx. 50 kDa) was present on the resin together with the bait GST-FL (lane ‘GST-FL/P’). Input (I) shows the untagged FL in solution. Recombinant immobilised GST was used as a negative control (lane ‘GST/P’). C) Western blot analysis (anti-testin (green), anti-GST (red)) of a mock buffer control incubated with immobilised GST-FL on resin. Similar as in B, the buffer contains inactivated thrombin but no soluble FL prey. Untagged FL is absent on the resin (P) (compare to Fig 2B , lane ‘GST-FL/P’) indicating that possible residual thrombin activity is not cleaving the GST-FL on the resin. D, E, F) Immobilised recombinant GST-FL (bait) was incubated with soluble untagged Evl (positive control, D) or cofilin (negative control, F) as preys. Immobilised GST was incubated with Evl (prey) and used as additional negative control (E). Western blot analysis of inputs (I) and proteins on the resin (P) is shown using anti testin (green), anti-Evl, anti-cofilin and anti-GST (red) antibodies. Untagged Evl (prey) is present on the GST-FL resin (lane P, 2D). Positions of bait, prey and negative control bait (Neg Ctr) are indicated in each panel. M: marker proteins (kDa).

Techniques Used: In Vitro, Affinity Purification, Recombinant, Western Blot, Incubation, Negative Control, Activity Assay, Positive Control, Marker

45) Product Images from "Y-box binding protein 1 enhances DNA topoisomerase 1 activity and sensitivity to camptothecin via direct interaction"

Article Title: Y-box binding protein 1 enhances DNA topoisomerase 1 activity and sensitivity to camptothecin via direct interaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-014-0112-7

YB-1 promotes TOPO1 activity in DNA relaxation assays. A . Purification of recombinant proteins for DNA relaxation assays. Full-length YB-1 or YB-1 deletion mutants were expressed in bacterial cells, purified with 15 μl of glutathione-Sepharose 4B, and subjected to SDS-PAGE and Coomassie blue staining. B . Recombinant YB-1 promotes relaxation of supercoiled DNA. pGEM-T easy supercoiled DNA (0.25 μg) was incubated with TOPO1 (1 ng), GST, GST-YB-1 (40 or 400 ng) protein, or a combination of TOPO1 and GST-YB-1 for 30 minutes at 37°C (left panel). To test which part of YB-1 contained the TOPO1-binding domain, pGEM-T easy supercoiled DNA (0.25 μg) was incubated with TOPO1 (1 ng) alone, and TOPO1 (1 ng) with either GST, GST-YB-1, GST-YB-1 Δ1, GST-YB-1 Δ2, GST-YB-1 Δ3, or GST (400 ng) protein, for 30 minutes at 37°C (right panel). The DNA was resolved on agarose gels (without ethidium bromide), and stained thereafter with ethidium bromide. The supercoiled (sc) and relaxed (r) DNA bands are shown. C . Endogenous YB-1 knockdown with siRNA reduces TOPO1 DNA relaxation activity. PC-3 cells were transiently transfected with human YB-1 siRNA or control siRNA, and nuclear extracts (50 μg) were subjected to SDS–PAGE and western blotting. Transferred proteins were probed with anti-YB-1 and anti-TOPO1 antibodies, using anti-laminB1 antibodies as a loading control for nuclear protein (left panel). Forty-eight hours after the aforementioned transfection, various amounts of PC-3 nuclear extracts (NE) (1 μg to 4 μg of NE prepared by dilution in phosphate-buffered saline) were incubated with pGEM-T easy supercoiled DNA (0.25 μg) for 30 minutes at 37°C. The supercoiled and relaxed DNA bands are shown in the right panel.
Figure Legend Snippet: YB-1 promotes TOPO1 activity in DNA relaxation assays. A . Purification of recombinant proteins for DNA relaxation assays. Full-length YB-1 or YB-1 deletion mutants were expressed in bacterial cells, purified with 15 μl of glutathione-Sepharose 4B, and subjected to SDS-PAGE and Coomassie blue staining. B . Recombinant YB-1 promotes relaxation of supercoiled DNA. pGEM-T easy supercoiled DNA (0.25 μg) was incubated with TOPO1 (1 ng), GST, GST-YB-1 (40 or 400 ng) protein, or a combination of TOPO1 and GST-YB-1 for 30 minutes at 37°C (left panel). To test which part of YB-1 contained the TOPO1-binding domain, pGEM-T easy supercoiled DNA (0.25 μg) was incubated with TOPO1 (1 ng) alone, and TOPO1 (1 ng) with either GST, GST-YB-1, GST-YB-1 Δ1, GST-YB-1 Δ2, GST-YB-1 Δ3, or GST (400 ng) protein, for 30 minutes at 37°C (right panel). The DNA was resolved on agarose gels (without ethidium bromide), and stained thereafter with ethidium bromide. The supercoiled (sc) and relaxed (r) DNA bands are shown. C . Endogenous YB-1 knockdown with siRNA reduces TOPO1 DNA relaxation activity. PC-3 cells were transiently transfected with human YB-1 siRNA or control siRNA, and nuclear extracts (50 μg) were subjected to SDS–PAGE and western blotting. Transferred proteins were probed with anti-YB-1 and anti-TOPO1 antibodies, using anti-laminB1 antibodies as a loading control for nuclear protein (left panel). Forty-eight hours after the aforementioned transfection, various amounts of PC-3 nuclear extracts (NE) (1 μg to 4 μg of NE prepared by dilution in phosphate-buffered saline) were incubated with pGEM-T easy supercoiled DNA (0.25 μg) for 30 minutes at 37°C. The supercoiled and relaxed DNA bands are shown in the right panel.

Techniques Used: Activity Assay, Purification, Recombinant, SDS Page, Staining, Incubation, Binding Assay, Transfection, Western Blot

Identification of the TOPO1 binding domain in YB-1. A . Schematic illustration of the GST-YB-1 deletion mutants (Ise et al., 1999 [ 26 ]). CSD indicates the cold shock domain. Amino acid residues are numbered. B . Interaction of GST-YB-1 deletion mutants with ThioHis-TOPO1. Approximately 500 μg of each GST fusion protein was immobilized on 15 μl of glutathione-Sepharose 4B, and the resin was incubated with 500 μg of ThioHis-TOPO1. Resin bound proteins were examined by western blotting using anti-Thio (upper panel) or anti-GST (lower panel) antibodies. C . Interaction of GST-YB-1 deletion mutants with TOPO1 in nuclear lysis solutions of PC-3 cells. Approximately 500 μg of each GST fusion protein was immobilized on 15 μl of glutathione-Sepharose 4B, and the resin was incubated with 500 μg of PC-3 nuclear lysis. Resin bound proteins were western blotted and the membrane probed using TOPO1 (upper panel) or anti-GST (lower panel) antibodies.
Figure Legend Snippet: Identification of the TOPO1 binding domain in YB-1. A . Schematic illustration of the GST-YB-1 deletion mutants (Ise et al., 1999 [ 26 ]). CSD indicates the cold shock domain. Amino acid residues are numbered. B . Interaction of GST-YB-1 deletion mutants with ThioHis-TOPO1. Approximately 500 μg of each GST fusion protein was immobilized on 15 μl of glutathione-Sepharose 4B, and the resin was incubated with 500 μg of ThioHis-TOPO1. Resin bound proteins were examined by western blotting using anti-Thio (upper panel) or anti-GST (lower panel) antibodies. C . Interaction of GST-YB-1 deletion mutants with TOPO1 in nuclear lysis solutions of PC-3 cells. Approximately 500 μg of each GST fusion protein was immobilized on 15 μl of glutathione-Sepharose 4B, and the resin was incubated with 500 μg of PC-3 nuclear lysis. Resin bound proteins were western blotted and the membrane probed using TOPO1 (upper panel) or anti-GST (lower panel) antibodies.

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

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

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002833

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

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

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

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

47) Product Images from "Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells"

Article Title: Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00061-17

The PBD mutant facilitates PP1α-MEK2 interaction less efficiently than full-length mortalin. (A and B) Dose-dependent effects of mortalin on MEK2-PP1α interaction. Recombinant MEK2 and PP1α (0.5 μM) were incubated for 60 min in the presence of different doses of GST-mortalin (A) or GST-PBD (B) before IP using anti-MEK2 antibody and protein G-agarose. (C) The degree of PP1α-MEK2 interaction affected by GST-mortalin or GST-PBD was determined by densitometry of the PP1α signals normalized by MEK2 signals in panels A and B.
Figure Legend Snippet: The PBD mutant facilitates PP1α-MEK2 interaction less efficiently than full-length mortalin. (A and B) Dose-dependent effects of mortalin on MEK2-PP1α interaction. Recombinant MEK2 and PP1α (0.5 μM) were incubated for 60 min in the presence of different doses of GST-mortalin (A) or GST-PBD (B) before IP using anti-MEK2 antibody and protein G-agarose. (C) The degree of PP1α-MEK2 interaction affected by GST-mortalin or GST-PBD was determined by densitometry of the PP1α signals normalized by MEK2 signals in panels A and B.

Techniques Used: Mutagenesis, Recombinant, Incubation

Effects of adenosine nucleotides on mortalin interactions with PP1α and MEK2. GST-mortalin (A and B) or GST-PBD (C and D) (0.5 μM) was incubated for 60 min with 0.5 μM recombinant PP1α (A and C) or MEK2 (B and D) in the presence of 1 μM ATP or ADP before GST pulldown using glutathione-Sepharose and Western blotting.
Figure Legend Snippet: Effects of adenosine nucleotides on mortalin interactions with PP1α and MEK2. GST-mortalin (A and B) or GST-PBD (C and D) (0.5 μM) was incubated for 60 min with 0.5 μM recombinant PP1α (A and C) or MEK2 (B and D) in the presence of 1 μM ATP or ADP before GST pulldown using glutathione-Sepharose and Western blotting.

Techniques Used: Incubation, Recombinant, Western Blot

48) Product Images from "Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA"

Article Title: Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA

Journal: Journal of Cell Science

doi: 10.1242/jcs.056176

Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or GST-KSRP-Sepharose beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P
Figure Legend Snippet: Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or GST-KSRP-Sepharose beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P

Techniques Used: Transfection, Lysis, Incubation, In Vitro, Synthesized, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

KSRP interacts with Dvl3. ( A ) F9 cells were transiently transfected with FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. Asterisks indicate the bands of immunoglobulin heavy and light chains. ( B ) F9 cells were treated with 100 nM of Dvl3 siRNA for 24 hours followed by transient expression of FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. ( C ) F9 cell lysates were immunoprecipitated with either rabbit control IgG or rabbit anti-KSRP polyclonal antibody and the interaction of KSRP with Dvl3 was visualized by probing the blots with anti-Dvl3 mouse monoclonal antibody. ( D ) F9 cells were transiently transfected with empty vector or FLAG-KSRP for 24 hours. The cells were then treated with Wnt3a (10 ng/ml) for indicated period of time followed by cell lysis and affinity pull-downs with anti-Dvl3 specific antibodies followed by immunoblotting with anti-FLAG antibodies. ( E ) To test the direct interaction of Dvl3 with KSRP, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST- or GST-KSRP-Sepharose beads in the presence of 0.8% BSA. The interaction was visualized by SDS-PAGE and autoradiography. Representative blots of three independent experiments that proved highly reproducible are shown. * P
Figure Legend Snippet: KSRP interacts with Dvl3. ( A ) F9 cells were transiently transfected with FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. Asterisks indicate the bands of immunoglobulin heavy and light chains. ( B ) F9 cells were treated with 100 nM of Dvl3 siRNA for 24 hours followed by transient expression of FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. ( C ) F9 cell lysates were immunoprecipitated with either rabbit control IgG or rabbit anti-KSRP polyclonal antibody and the interaction of KSRP with Dvl3 was visualized by probing the blots with anti-Dvl3 mouse monoclonal antibody. ( D ) F9 cells were transiently transfected with empty vector or FLAG-KSRP for 24 hours. The cells were then treated with Wnt3a (10 ng/ml) for indicated period of time followed by cell lysis and affinity pull-downs with anti-Dvl3 specific antibodies followed by immunoblotting with anti-FLAG antibodies. ( E ) To test the direct interaction of Dvl3 with KSRP, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST- or GST-KSRP-Sepharose beads in the presence of 0.8% BSA. The interaction was visualized by SDS-PAGE and autoradiography. Representative blots of three independent experiments that proved highly reproducible are shown. * P

Techniques Used: Transfection, Lysis, Expressing, Immunoprecipitation, Plasmid Preparation, In Vitro, Synthesized, Labeling, SDS Page, Autoradiography

49) Product Images from "Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation"

Article Title: Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201205170

Mad2 prevents Cdc20 from interacting with the APC/C. (A) The hydrophobic core of the KILR motif is essential to bind Mad2. E. coli extracts expressing GST, GST fused to the N terminus of wild-type Cdc20, or the indicated mutants were incubated with recombinant human Mad2 for 30 min at 4°C and purified with glutathione–Sepharose, and the amount of Mad2 was analyzed by quantitative immunoblotting. Relative amount of Mad2 bound is shown at the bottom. Results are representative of three independent experiments. Asterisk shows a truncated form of Cdc20 that does not bind Mad2. (B) The IL motif is required to bind to the APC/C. In vitro translated (IVT) full-length 3×Flag–wild-type Cdc20 or the indicated mutants were analyzed as in Fig. 2 C . Results are representative of three independent experiments. (C) The KILR and C box motifs are required for the N terminus of Cdc20 to interact with the APC/C. GST fusion proteins of the N terminus of Cdc20 (N151), wild type, and the indicated mutants were incubated for 40 min at 4°C with mitotic extracts depleted of endogenous Cdc20. Proteins retained on the beads were analyzed by quantitative immunoblotting with the indicated antibodies. Results are representative of three independent experiments. (D and E) Mad2 competes with the APC/C for binding to the KILR motif. (D) GST or GST fusion proteins of the N terminus of wild-type Cdc20 or the ΔKILR mutant were incubated with recombinant Mad2 and mitotic HeLa cell extracts as in C. The APC/C bound to the beads was analyzed by quantitative immunoblotting with the indicated antibodies (Western blot [WB]). Recombinant proteins were also detected by Coomassie blue staining (CBB). (E) GST or GST fusion proteins were prebound to gluthatione–Sepharose and incubated with mitotic extract plus recombinant Mad2. “Pre” indicates the fusion protein was incubated with Mad2 before the mitotic extract. Mad2 was added in fourfold excess over the amount of GST-N151. Samples were analyzed by quantitative immunoblotting with the indicated antibodies. The relative amount of the APC/C and Mad2 bound to the beads is shown on the bottom where the amount of APC/C bound at 40 min or the amount of Mad2 bound at 40 min in the “pre” sample is set to 1. Binding assays in D and E are representative of three experiments. end, endogenous; IP, immunoprecipitation; WT, wild type.
Figure Legend Snippet: Mad2 prevents Cdc20 from interacting with the APC/C. (A) The hydrophobic core of the KILR motif is essential to bind Mad2. E. coli extracts expressing GST, GST fused to the N terminus of wild-type Cdc20, or the indicated mutants were incubated with recombinant human Mad2 for 30 min at 4°C and purified with glutathione–Sepharose, and the amount of Mad2 was analyzed by quantitative immunoblotting. Relative amount of Mad2 bound is shown at the bottom. Results are representative of three independent experiments. Asterisk shows a truncated form of Cdc20 that does not bind Mad2. (B) The IL motif is required to bind to the APC/C. In vitro translated (IVT) full-length 3×Flag–wild-type Cdc20 or the indicated mutants were analyzed as in Fig. 2 C . Results are representative of three independent experiments. (C) The KILR and C box motifs are required for the N terminus of Cdc20 to interact with the APC/C. GST fusion proteins of the N terminus of Cdc20 (N151), wild type, and the indicated mutants were incubated for 40 min at 4°C with mitotic extracts depleted of endogenous Cdc20. Proteins retained on the beads were analyzed by quantitative immunoblotting with the indicated antibodies. Results are representative of three independent experiments. (D and E) Mad2 competes with the APC/C for binding to the KILR motif. (D) GST or GST fusion proteins of the N terminus of wild-type Cdc20 or the ΔKILR mutant were incubated with recombinant Mad2 and mitotic HeLa cell extracts as in C. The APC/C bound to the beads was analyzed by quantitative immunoblotting with the indicated antibodies (Western blot [WB]). Recombinant proteins were also detected by Coomassie blue staining (CBB). (E) GST or GST fusion proteins were prebound to gluthatione–Sepharose and incubated with mitotic extract plus recombinant Mad2. “Pre” indicates the fusion protein was incubated with Mad2 before the mitotic extract. Mad2 was added in fourfold excess over the amount of GST-N151. Samples were analyzed by quantitative immunoblotting with the indicated antibodies. The relative amount of the APC/C and Mad2 bound to the beads is shown on the bottom where the amount of APC/C bound at 40 min or the amount of Mad2 bound at 40 min in the “pre” sample is set to 1. Binding assays in D and E are representative of three experiments. end, endogenous; IP, immunoprecipitation; WT, wild type.

Techniques Used: Cytotoxicity Assay, Expressing, Incubation, Recombinant, Purification, In Vitro, Binding Assay, Mutagenesis, Western Blot, Staining, Immunoprecipitation

The ΔKILR motif interacts with the APC/C in a different manner compared with the IR tail and the C box. (A and B) The IR tail and C box motif in Cdc20 are required to interact with the APC/C. (A) HeLa cells expressing 3×Flag–wild-type, ΔIR, or C box mutant Cdc20 from an inducible promoter were treated with siRNA against Cdc20 and synchronized at mitosis as in Fig. 2 B . The APC/C was immunoprecipitated with anti-APC4 antibodies and analyzed by quantitative immunoblotting. Results are representative of three independent experiments. (B) In vitro translated (IVT) full-length 3×Flag–wild type, ΔIR, or C box mutants of Cdc20 were tested for their ability to bind to the APC/C as in Fig. 2 C . Results are representative of four independent experiments. (C and D) The ΔIR and C box mutants of Cdc20 can still interact with the APC/C when part of the MCC. (C) HeLa cell line expressing inducible 3×Flag-tagged wild-type, ΔIR, C box, or ΔKILR mutant Cdc20 were treated with siRNA against Cdc20, arrested at prometaphase with 0.33 µM nocodazole, and harvested by mitotic shake off. The APC/C was immunoprecipitated using anti-APC4 antibodies and analyzed by quantitative immunoblotting. Results are representative of three independent experiments. (D) HeLa cell lines expressing inducible 3×Flag-tagged wild-type or ΔIR mutant Cdc20 were treated with siRNA against Cdc20 or Cdc20 and APC3, arrested at prometaphase, and analyzed as in C. (E and F) The ΔKILR mutant is not able to form the MCC or bind to the APC/C. (E) HeLa cell lines expressing inducible 3×Flag-Cdc20 wt or Cdc20 ΔKILR were treated with siRNA against Cdc20, arrested in prometaphase with 0.33 µM nocodazole, and harvested by mitotic shake off. Extracts were analyzed by size-exclusion chromatography on a Sepharose 6 column, and fractions were analyzed by quantitative immunoblotting with antibodies against Cdc20. The peaks of APC/C and MCC migration are indicated. Results are representative of two independent experiments. (F) Distributions of wild-type Cdc20 and ΔKILR mutant with the sum of Cdc20 intensities set to 1. Immunoblotting with antibodies against APC3, Cdc20, BubR1, and Mad2 is shown in Fig. S3 . end, endogenous; IP, immunoprecipitation; WT, wild type.
Figure Legend Snippet: The ΔKILR motif interacts with the APC/C in a different manner compared with the IR tail and the C box. (A and B) The IR tail and C box motif in Cdc20 are required to interact with the APC/C. (A) HeLa cells expressing 3×Flag–wild-type, ΔIR, or C box mutant Cdc20 from an inducible promoter were treated with siRNA against Cdc20 and synchronized at mitosis as in Fig. 2 B . The APC/C was immunoprecipitated with anti-APC4 antibodies and analyzed by quantitative immunoblotting. Results are representative of three independent experiments. (B) In vitro translated (IVT) full-length 3×Flag–wild type, ΔIR, or C box mutants of Cdc20 were tested for their ability to bind to the APC/C as in Fig. 2 C . Results are representative of four independent experiments. (C and D) The ΔIR and C box mutants of Cdc20 can still interact with the APC/C when part of the MCC. (C) HeLa cell line expressing inducible 3×Flag-tagged wild-type, ΔIR, C box, or ΔKILR mutant Cdc20 were treated with siRNA against Cdc20, arrested at prometaphase with 0.33 µM nocodazole, and harvested by mitotic shake off. The APC/C was immunoprecipitated using anti-APC4 antibodies and analyzed by quantitative immunoblotting. Results are representative of three independent experiments. (D) HeLa cell lines expressing inducible 3×Flag-tagged wild-type or ΔIR mutant Cdc20 were treated with siRNA against Cdc20 or Cdc20 and APC3, arrested at prometaphase, and analyzed as in C. (E and F) The ΔKILR mutant is not able to form the MCC or bind to the APC/C. (E) HeLa cell lines expressing inducible 3×Flag-Cdc20 wt or Cdc20 ΔKILR were treated with siRNA against Cdc20, arrested in prometaphase with 0.33 µM nocodazole, and harvested by mitotic shake off. Extracts were analyzed by size-exclusion chromatography on a Sepharose 6 column, and fractions were analyzed by quantitative immunoblotting with antibodies against Cdc20. The peaks of APC/C and MCC migration are indicated. Results are representative of two independent experiments. (F) Distributions of wild-type Cdc20 and ΔKILR mutant with the sum of Cdc20 intensities set to 1. Immunoblotting with antibodies against APC3, Cdc20, BubR1, and Mad2 is shown in Fig. S3 . end, endogenous; IP, immunoprecipitation; WT, wild type.

Techniques Used: Expressing, Mutagenesis, Immunoprecipitation, In Vitro, Size-exclusion Chromatography, Migration

50) Product Images from "E3 Ubiquitin Ligase RNF31 Cooperates with DAX-1 in Transcriptional Repression of Steroidogenesis ▿E3 Ubiquitin Ligase RNF31 Cooperates with DAX-1 in Transcriptional Repression of Steroidogenesis ▿ †"

Article Title: E3 Ubiquitin Ligase RNF31 Cooperates with DAX-1 in Transcriptional Repression of Steroidogenesis ▿E3 Ubiquitin Ligase RNF31 Cooperates with DAX-1 in Transcriptional Repression of Steroidogenesis ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00743-08

RNF31 associates with DAX-1. (A) Domain structure of human RNF31 (aa 1 to 1072). PUB, putative AAA ATPase-binding domain; ZnF_RBZ, putative zinc-finger ubiquitin-binding domain; RING-IBR-RING, E3 ubiquitin ligase domain. (B) Domain structures of human DAX-1 (WT, aa 1 to 470) and derivatives used for interaction assays. N, N-terminal domain consisting of three repeats (R1 to R3); C, C-terminal repressor domain homologous to the NR LBD; R3, third repeat region. (C) Gal4-DAX-1 WT, N terminus, or C terminus constructs were tested for interaction with GAD-RNF31 variants in a yeast two-hybrid liquid β-galactosidase assay. Relative β-galactosidase units represent absolute units relative to the value for the GAD negative control (Gal4 activation domain only). The expression of RNF31 constructs was verified by Western blotting (see Fig. S1 in the supplemental material). (D) COS-7 cells were cotransfected with DAX-1, Flag-RNF31, or Flag as indicated. Whole-cell extracts were subjected to immunoprecipitation (IP) using a rabbit polyclonal anti-Flag (αFlag) antibody and analyzed by Western blotting with anti-DAX-1 (αDAX) and anti-Flag mouse monoclonal antibodies. (E) Glutathione-Sepharose-bound GST-DAX-1 constructs and GST alone were probed for interaction with in vitro-translated 35 S-labeled RNF31 in a GST pull-down assay. (F) pGAD-RNF31 was tested for interaction with various NRs by using a yeast two-hybrid liquid β-galactosidase assay. Relative β-galactosidase units represent absolute units relative to the value for the GBT9 negative control (empty vector). ERα, estrogen receptor α.
Figure Legend Snippet: RNF31 associates with DAX-1. (A) Domain structure of human RNF31 (aa 1 to 1072). PUB, putative AAA ATPase-binding domain; ZnF_RBZ, putative zinc-finger ubiquitin-binding domain; RING-IBR-RING, E3 ubiquitin ligase domain. (B) Domain structures of human DAX-1 (WT, aa 1 to 470) and derivatives used for interaction assays. N, N-terminal domain consisting of three repeats (R1 to R3); C, C-terminal repressor domain homologous to the NR LBD; R3, third repeat region. (C) Gal4-DAX-1 WT, N terminus, or C terminus constructs were tested for interaction with GAD-RNF31 variants in a yeast two-hybrid liquid β-galactosidase assay. Relative β-galactosidase units represent absolute units relative to the value for the GAD negative control (Gal4 activation domain only). The expression of RNF31 constructs was verified by Western blotting (see Fig. S1 in the supplemental material). (D) COS-7 cells were cotransfected with DAX-1, Flag-RNF31, or Flag as indicated. Whole-cell extracts were subjected to immunoprecipitation (IP) using a rabbit polyclonal anti-Flag (αFlag) antibody and analyzed by Western blotting with anti-DAX-1 (αDAX) and anti-Flag mouse monoclonal antibodies. (E) Glutathione-Sepharose-bound GST-DAX-1 constructs and GST alone were probed for interaction with in vitro-translated 35 S-labeled RNF31 in a GST pull-down assay. (F) pGAD-RNF31 was tested for interaction with various NRs by using a yeast two-hybrid liquid β-galactosidase assay. Relative β-galactosidase units represent absolute units relative to the value for the GBT9 negative control (empty vector). ERα, estrogen receptor α.

Techniques Used: Binding Assay, Construct, Negative Control, Activation Assay, Expressing, Western Blot, Immunoprecipitation, In Vitro, Labeling, Pull Down Assay, Plasmid Preparation

RNF31 is necessary for DAX-1 corepressor complex recruitment and stability at the human StAR and CYP19 promoters. (A) H295R cells treated for 72 h with siLUC, siSF-1, or siRNF31 were subjected to ChiP using the indicated antibodies. Precipitated DNA was analyzed with conventional PCR (agarose gel) and qPCR (graph) to determine the recruitment of transcription factors and coregulators to StAR (left) and CYP19 (right) promoters. qPCR data are presented as changes relative to the siLUC (control) value, which was set to 1. IgG, immunoglobulin G. (B) Proposed model of the role of RNF31 in corepressor complex assembly (repressed promoters; left) and exchange upon activation of steroidogenic transcription (active promoters; right). Ub, ubiquitin; HDAC, histone deacetylase; GTFs, general transcription factors; SFRE, SF-1 response element.
Figure Legend Snippet: RNF31 is necessary for DAX-1 corepressor complex recruitment and stability at the human StAR and CYP19 promoters. (A) H295R cells treated for 72 h with siLUC, siSF-1, or siRNF31 were subjected to ChiP using the indicated antibodies. Precipitated DNA was analyzed with conventional PCR (agarose gel) and qPCR (graph) to determine the recruitment of transcription factors and coregulators to StAR (left) and CYP19 (right) promoters. qPCR data are presented as changes relative to the siLUC (control) value, which was set to 1. IgG, immunoglobulin G. (B) Proposed model of the role of RNF31 in corepressor complex assembly (repressed promoters; left) and exchange upon activation of steroidogenic transcription (active promoters; right). Ub, ubiquitin; HDAC, histone deacetylase; GTFs, general transcription factors; SFRE, SF-1 response element.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Activation Assay, Histone Deacetylase Assay

51) Product Images from "Identification of SFBB-Containing Canonical and Noncanonical SCF Complexes in Pollen of Apple (Malus x domestica)"

Article Title: Identification of SFBB-Containing Canonical and Noncanonical SCF Complexes in Pollen of Apple (Malus x domestica)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097642

In vitro binding assays of MdSSK1 and MdSBP1 with MdCUL1s. The interactions of MdCUL1s with MdSSK1 (A) and MdSBP1 (B) were tested. GST: MdSSK1, GST: MdSBP1 and GST (negative control) were expressed in E. coli and reacted with Glutathione Sepharose 4B. These sepharose bound recombinant proteins were incubated with MdCUL1A: FLAG and MdCUL1B: FLAG expressed in a cell-free system. Eluted proteins were separated by SDS-PAGE and detected by using an anti-FLAG antibody (top). Protein loading was checked by Ponceau-S staining of the blot before immunoblotting (bottom). Single and double asterisks indicate the GST-fusion protein and GST, respectively.
Figure Legend Snippet: In vitro binding assays of MdSSK1 and MdSBP1 with MdCUL1s. The interactions of MdCUL1s with MdSSK1 (A) and MdSBP1 (B) were tested. GST: MdSSK1, GST: MdSBP1 and GST (negative control) were expressed in E. coli and reacted with Glutathione Sepharose 4B. These sepharose bound recombinant proteins were incubated with MdCUL1A: FLAG and MdCUL1B: FLAG expressed in a cell-free system. Eluted proteins were separated by SDS-PAGE and detected by using an anti-FLAG antibody (top). Protein loading was checked by Ponceau-S staining of the blot before immunoblotting (bottom). Single and double asterisks indicate the GST-fusion protein and GST, respectively.

Techniques Used: In Vitro, Binding Assay, Negative Control, Recombinant, Incubation, SDS Page, Staining

In vitro binding assays of MdSSK1 and MdSBP1 with MdSFBB1- S 9 and MdSFBB1- S 9 -N. (A) Interactions of MdSSK1 and MdSBP1 with MdSFBB1- S 9 and MdSFBB1- S 9 -N. MBP: MdSSK1, MBP: MdSBP1 and MBP (negative control) were reacted with amylose resin. These beads bound recombinant proteins were incubated with GST: MdSFBB1- S 9 : FLAG and GST: MdSFBB1- S 9 -N: FLAG. Eluted proteins were separated by SDS-PAGE and detected using an anti-FLAG antibody (top). Protein loading was checked by Ponceau-S staining of the blot before immunoblotting (bottom). Single, double and triple asterisks, indicate MBP: MdSBP1, MBP: MdSSK1 and MBP, respectively. Diamonds indicate non-specific signals. (B) Competitive pull-down assay of MdSFBB1- S 9 and MdSFBB1- S 9 -N with MdSSK1 and MdSBP1. GST: MdSFBB1- S 9 : FLAG, GST: MdSFBB1- S 9 -N: FLAG and GST (negative control) were reacted with Glutathione Sepharose 4B. These sepharose bound recombinant proteins were incubated with an equal amount protein mixture of MBP: MdSSK1 (15 µg) and MBP: MdSBP1 (15 µg). Eluted proteins were separated by SDS-PAGE and detected using an anti-MBP antibody (top). Protein loading was checked by Ponceau-S staining of the blot before immunoblotting (bottom). Single, double and triple asterisks, indicate GST: MdSFBB1- S 9 : FLAG, GST: MdSFBB1- S 9 -N: FLAG and GST, respectively. Opened and closed arrows indicate MBP: MdSBP1 and MBP: MdSSK1, respectively. Diamonds indicate the probable truncated GST: MdSFBB1- S 9 : FLAG. (C) Competitive pull-down assay of MdSSK1 and MdSBP1 with MdSFBB1- S 9 and MdSFBB1- S 9 -N. MBP: MdSSK1, MBP: MdSBP1 and MBP (negative control) were reacted with amylose resin. These beads bound recombinant proteins were incubated with a protein mixture of approximately equal molecular numbers of GST: MdSFBB1- S 9 : FLAG (74 kDa, 4.5 µg) and GST: MdSFBB1- S 9 -N: FLAG (35 kDa, 2.1 µg). Eluted proteins were separated by SDS-PAGE and detected using an anti-FLAG antibody (top). Protein loading was checked by Ponceau-S staining of the blot before immunoblotting (bottom). Single, double and triple asterisks, indicate MBP: MdSBP1, MBP: MdSSK1 and MBP, respectively. Opened and closed triangles indicate specific GST: MdSFBB1- S 9 : FLAG and GST: MdSFBB1- S 9 -N: FLAG signals, respectively.
Figure Legend Snippet: In vitro binding assays of MdSSK1 and MdSBP1 with MdSFBB1- S 9 and MdSFBB1- S 9 -N. (A) Interactions of MdSSK1 and MdSBP1 with MdSFBB1- S 9 and MdSFBB1- S 9 -N. MBP: MdSSK1, MBP: MdSBP1 and MBP (negative control) were reacted with amylose resin. These beads bound recombinant proteins were incubated with GST: MdSFBB1- S 9 : FLAG and GST: MdSFBB1- S 9 -N: FLAG. Eluted proteins were separated by SDS-PAGE and detected using an anti-FLAG antibody (top). Protein loading was checked by Ponceau-S staining of the blot before immunoblotting (bottom). Single, double and triple asterisks, indicate MBP: MdSBP1, MBP: MdSSK1 and MBP, respectively. Diamonds indicate non-specific signals. (B) Competitive pull-down assay of MdSFBB1- S 9 and MdSFBB1- S 9 -N with MdSSK1 and MdSBP1. GST: MdSFBB1- S 9 : FLAG, GST: MdSFBB1- S 9 -N: FLAG and GST (negative control) were reacted with Glutathione Sepharose 4B. These sepharose bound recombinant proteins were incubated with an equal amount protein mixture of MBP: MdSSK1 (15 µg) and MBP: MdSBP1 (15 µg). Eluted proteins were separated by SDS-PAGE and detected using an anti-MBP antibody (top). Protein loading was checked by Ponceau-S staining of the blot before immunoblotting (bottom). Single, double and triple asterisks, indicate GST: MdSFBB1- S 9 : FLAG, GST: MdSFBB1- S 9 -N: FLAG and GST, respectively. Opened and closed arrows indicate MBP: MdSBP1 and MBP: MdSSK1, respectively. Diamonds indicate the probable truncated GST: MdSFBB1- S 9 : FLAG. (C) Competitive pull-down assay of MdSSK1 and MdSBP1 with MdSFBB1- S 9 and MdSFBB1- S 9 -N. MBP: MdSSK1, MBP: MdSBP1 and MBP (negative control) were reacted with amylose resin. These beads bound recombinant proteins were incubated with a protein mixture of approximately equal molecular numbers of GST: MdSFBB1- S 9 : FLAG (74 kDa, 4.5 µg) and GST: MdSFBB1- S 9 -N: FLAG (35 kDa, 2.1 µg). Eluted proteins were separated by SDS-PAGE and detected using an anti-FLAG antibody (top). Protein loading was checked by Ponceau-S staining of the blot before immunoblotting (bottom). Single, double and triple asterisks, indicate MBP: MdSBP1, MBP: MdSSK1 and MBP, respectively. Opened and closed triangles indicate specific GST: MdSFBB1- S 9 : FLAG and GST: MdSFBB1- S 9 -N: FLAG signals, respectively.

Techniques Used: In Vitro, Binding Assay, Negative Control, Recombinant, Incubation, SDS Page, Staining, Pull Down Assay

52) 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

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

53) Product Images from "The Zinc-Finger Transcription Factor INSM1 Is Expressed during Embryo Development and Interacts with the Cbl-Associated Protein"

Article Title: The Zinc-Finger Transcription Factor INSM1 Is Expressed during Embryo Development and Interacts with the Cbl-Associated Protein

Journal: Genomics

doi:

Mouse INSM1 interacts with CAP in vitro . (A) Expressed and purified GST (lane 1) and GST–CAP (lane 2) fusion proteins were electrophoresed on a 12% SDS-PAGE and stained by Coomassie blue. (B) The GST–CAP fusion protein was immobilized on glutathione Sepharose beads and then incubated with 35 S-labeled INSM1. After extensive washing, bound [ 35 S]INSM1 was eluted and separated on a 12% SDS-PAGE. Lane 3 shows that the 58-kDa 35 S-labeled INSM1 protein interacted with and was pulled down by GST–CAP, but not by GST alone (lane 2). The first lane, loaded with 35 S-labeled INSM1 protein, served as a size control.
Figure Legend Snippet: Mouse INSM1 interacts with CAP in vitro . (A) Expressed and purified GST (lane 1) and GST–CAP (lane 2) fusion proteins were electrophoresed on a 12% SDS-PAGE and stained by Coomassie blue. (B) The GST–CAP fusion protein was immobilized on glutathione Sepharose beads and then incubated with 35 S-labeled INSM1. After extensive washing, bound [ 35 S]INSM1 was eluted and separated on a 12% SDS-PAGE. Lane 3 shows that the 58-kDa 35 S-labeled INSM1 protein interacted with and was pulled down by GST–CAP, but not by GST alone (lane 2). The first lane, loaded with 35 S-labeled INSM1 protein, served as a size control.

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

Co-immunoprecipitation of mouse INSM1 and CAP in cultured cells. (A) Expression of CAP tagged with c-myc (lanes 1 and 2) and mouse INSM1 tagged with GFP (lanes 4 and 5) in Calu-6 (lanes 1 and 4) and HeLa (lanes 2 and 5) cells. Lanes 3 and 6 represent untransfected cells. Cell extracts were separated on SDS-PAGE, transferred to a nitrocellulose membrane, and stained with antibody to c-myc or GFP. The 37-kDa and 78-kDa bands, respectively, show that CAP and INSM1 were strongly expressed in Calu-6 and HeLa cells. (B) Calu-6 and HeLa cells were co-transfected with c-myc–CAP and INSM1–GFP. Cell lysates were immunoprecipitated with antibody to c-myc coupled to Sepharose beads (or Sepharose beads alone), transferred to a nitrocellulose membrane, and stained with antibody to c-myc or GFP. Lanes 1 and 2 show that immunoprecipitation with antibody to c-myc pulled down c-myc–CAP and lanes 4 and 5 show that GFP–INSM1 was co-precipitated with c-myc–CAP. Lanes 3 and 6 represent untransfected cells. The 61- and 27-kDa bands in lanes 1 and 2 are the heavy and light chains of the antibody to c-myc used for immunoprecipitation. (C) Calu-6 cells that express endogenous INSM1 were transfected with c-myc–CAP. Cell lysates were immunoprecipitated with antibody to c-myc that had been coupled to Sepharose beads, electrophoresed on SDS-PAGE, transferred to a nitrocel-lulose membrane, and stained with antibody to INSM1. Endogenous INSM1 co-precipitated in the c-myc–CAP pull-down.
Figure Legend Snippet: Co-immunoprecipitation of mouse INSM1 and CAP in cultured cells. (A) Expression of CAP tagged with c-myc (lanes 1 and 2) and mouse INSM1 tagged with GFP (lanes 4 and 5) in Calu-6 (lanes 1 and 4) and HeLa (lanes 2 and 5) cells. Lanes 3 and 6 represent untransfected cells. Cell extracts were separated on SDS-PAGE, transferred to a nitrocellulose membrane, and stained with antibody to c-myc or GFP. The 37-kDa and 78-kDa bands, respectively, show that CAP and INSM1 were strongly expressed in Calu-6 and HeLa cells. (B) Calu-6 and HeLa cells were co-transfected with c-myc–CAP and INSM1–GFP. Cell lysates were immunoprecipitated with antibody to c-myc coupled to Sepharose beads (or Sepharose beads alone), transferred to a nitrocellulose membrane, and stained with antibody to c-myc or GFP. Lanes 1 and 2 show that immunoprecipitation with antibody to c-myc pulled down c-myc–CAP and lanes 4 and 5 show that GFP–INSM1 was co-precipitated with c-myc–CAP. Lanes 3 and 6 represent untransfected cells. The 61- and 27-kDa bands in lanes 1 and 2 are the heavy and light chains of the antibody to c-myc used for immunoprecipitation. (C) Calu-6 cells that express endogenous INSM1 were transfected with c-myc–CAP. Cell lysates were immunoprecipitated with antibody to c-myc that had been coupled to Sepharose beads, electrophoresed on SDS-PAGE, transferred to a nitrocel-lulose membrane, and stained with antibody to INSM1. Endogenous INSM1 co-precipitated in the c-myc–CAP pull-down.

Techniques Used: Immunoprecipitation, Cell Culture, Expressing, SDS Page, Staining, Transfection

54) Product Images from "L290P/V mutations increase ERK3’s cytoplasmic localization and migration/invasion-promoting capability in cancer cells"

Article Title: L290P/V mutations increase ERK3’s cytoplasmic localization and migration/invasion-promoting capability in cancer cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-15135-9

L290P/V mutations do not alter ERK3 kinase activity. ( a ) Coomassie staining of purified wild type or mutant ERK3 proteins. 293 T cells were transfected with HA-tagged wild type ERK3, ERK3 L290P, L290V or kinase dead (KD) plasmids. ERK3 proteins were immunoprecipitated using HA-antibody-conjugated agarose beads, followed by elution with HA peptide. The purified proteins (300 ng) were analyzed by SDS-PAGE gel followed by Coomassie staining. The molecular size of each protein marker is indicated on the right side. ( b ) In vitro ERK3 kinase assay was performed by incubating 100 ng of purified ERK3 or each of ERK3 mutants as indicated, together with 1 μg of recombinant GST-SRC3-CID (substrate) in the presence of γ- 32 P-ATP. Phosphorylation of GST-SRC3-CID by ERK3 proteins was detected by autoradiograph (the right panel). Total protein level of GST-SRC3-CID in the reactions is shown by Coomassie staining (the left panel). Please note that ERK3 proteins are hardly seen in the Coomassie-stained gel due to their small amount (100 ng). ( c ) Quantification of GST-SRC3-CID phosphorylation by wild type or mutant ERK3 proteins. The relative phosphorylation level of GST-SRC3-CID is represented by the ratio of the band intensity of phosphorylated GST-SRC3-CID (shown in the autoradiograph) over that of the corresponding total GST-SRC3-CID (shown in the commassie-stained gel). For the purpose of comparison, the nomalized phosphorylation level of GST-SRC3-CID by wild type ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of 3 independent experiments. *P
Figure Legend Snippet: L290P/V mutations do not alter ERK3 kinase activity. ( a ) Coomassie staining of purified wild type or mutant ERK3 proteins. 293 T cells were transfected with HA-tagged wild type ERK3, ERK3 L290P, L290V or kinase dead (KD) plasmids. ERK3 proteins were immunoprecipitated using HA-antibody-conjugated agarose beads, followed by elution with HA peptide. The purified proteins (300 ng) were analyzed by SDS-PAGE gel followed by Coomassie staining. The molecular size of each protein marker is indicated on the right side. ( b ) In vitro ERK3 kinase assay was performed by incubating 100 ng of purified ERK3 or each of ERK3 mutants as indicated, together with 1 μg of recombinant GST-SRC3-CID (substrate) in the presence of γ- 32 P-ATP. Phosphorylation of GST-SRC3-CID by ERK3 proteins was detected by autoradiograph (the right panel). Total protein level of GST-SRC3-CID in the reactions is shown by Coomassie staining (the left panel). Please note that ERK3 proteins are hardly seen in the Coomassie-stained gel due to their small amount (100 ng). ( c ) Quantification of GST-SRC3-CID phosphorylation by wild type or mutant ERK3 proteins. The relative phosphorylation level of GST-SRC3-CID is represented by the ratio of the band intensity of phosphorylated GST-SRC3-CID (shown in the autoradiograph) over that of the corresponding total GST-SRC3-CID (shown in the commassie-stained gel). For the purpose of comparison, the nomalized phosphorylation level of GST-SRC3-CID by wild type ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of 3 independent experiments. *P

Techniques Used: Activity Assay, Staining, Purification, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Marker, In Vitro, Kinase Assay, Recombinant, Autoradiography

As compared to wild type ERK3, both L290P and L290V mutants have increased interactions with CRM1. HA-tagged wild type ERK3, ERK3 L290P or ERK3 L290V was exogenously expressed in HeLa cells. ERK3 protein complexes were immunoprecipitated using agarose beads conjugated with anti-HA antibodies, followed by Western blotting of the proteins as indicated in the figure. Input: 2% of the amount for immunoprecipitation (IP). Numbers below the immunoblots of CRM1 and MK5 in HA-IP samples represent the relative binding capacity of ERK3 (or L290P or V mutants) with these proteins, which is determined by the ratio of the band intensity in HA-IP over that in the corresponding input. For the purpose of comparison, the relative binding capacity of wild type ERK3 with either CRM1 or MK5 was arbitrarily set as 1.0.
Figure Legend Snippet: As compared to wild type ERK3, both L290P and L290V mutants have increased interactions with CRM1. HA-tagged wild type ERK3, ERK3 L290P or ERK3 L290V was exogenously expressed in HeLa cells. ERK3 protein complexes were immunoprecipitated using agarose beads conjugated with anti-HA antibodies, followed by Western blotting of the proteins as indicated in the figure. Input: 2% of the amount for immunoprecipitation (IP). Numbers below the immunoblots of CRM1 and MK5 in HA-IP samples represent the relative binding capacity of ERK3 (or L290P or V mutants) with these proteins, which is determined by the ratio of the band intensity in HA-IP over that in the corresponding input. For the purpose of comparison, the relative binding capacity of wild type ERK3 with either CRM1 or MK5 was arbitrarily set as 1.0.

Techniques Used: Immunoprecipitation, Western Blot, Binding Assay

55) Product Images from "Pitchfork and Gprasp2 Target Smoothened to the Primary Cilium for Hedgehog Pathway Activation"

Article Title: Pitchfork and Gprasp2 Target Smoothened to the Primary Cilium for Hedgehog Pathway Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0149477

Shh signaling induces Smo ciliary targeting complex formation. ( A ) Kinetics of Smo-Gprasp2-Pifo multimeric complex formation upon Shh treatment. Strep-Tactin sepharose-coupled mPifo was subjected to time-resolved affinity precipitations of native protein complexes formed upon Shh stimulation in PLCs. ( B ) Time-resolved endogenous co-IP of the Gprasp2-Smo ciliary targeting complex upon Shh stimulation in PLCs. Lysates from cells exposed to Shh for different time periods were subjected to immunoprecipitation with Anti-Smo antibody-conjugated beads. Immunoprecipitation with isotype-specific IgG antibodies served as negative control ( ** indicates non-specific bands). After immunoprecipitation, subsequent immunoblotting with the indicated antibodies determined dynamic changes in protein interactions (A, B). β-arrestin 1 and 2, well-known Smo interacting proteins, were used as a positive control. All error bars indicate the mean ± SD of three independent experiments. Data were analyzed using a two tailed unpaired t -test (* = p
Figure Legend Snippet: Shh signaling induces Smo ciliary targeting complex formation. ( A ) Kinetics of Smo-Gprasp2-Pifo multimeric complex formation upon Shh treatment. Strep-Tactin sepharose-coupled mPifo was subjected to time-resolved affinity precipitations of native protein complexes formed upon Shh stimulation in PLCs. ( B ) Time-resolved endogenous co-IP of the Gprasp2-Smo ciliary targeting complex upon Shh stimulation in PLCs. Lysates from cells exposed to Shh for different time periods were subjected to immunoprecipitation with Anti-Smo antibody-conjugated beads. Immunoprecipitation with isotype-specific IgG antibodies served as negative control ( ** indicates non-specific bands). After immunoprecipitation, subsequent immunoblotting with the indicated antibodies determined dynamic changes in protein interactions (A, B). β-arrestin 1 and 2, well-known Smo interacting proteins, were used as a positive control. All error bars indicate the mean ± SD of three independent experiments. Data were analyzed using a two tailed unpaired t -test (* = p

Techniques Used: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Positive Control, Two Tailed Test

Gprasp2 is required for ciliary localization of Smo and Shh target gene activation. ( A ) Shh-induced protein levels after 48h of siRNA-mediated knock-down of Gprasp2 in PLCs. ( B ) Mouse Gprasp2 siRNA target sequences with Multiple species ClustalW alignment to human and mouse Gprasp2 and indicated siRNA seed region position. Determination of Smo-Pifo complex formation after depletion of mGprasp2 ( C ) and rescue of mGprasp2 depletion by introducing hGPRASP2 ( D ). 48h post-transfection with the indicated siRNA duplexes or co-transfection with the indicated siRNA duplexes and HA-tagged hGPRASP2, PLCs were stimulated with Shh and the lysates were subsequently subjected to Strep-Tactin sepharose-coupled mPifo. Input (10%) and endogenous Smo protein complexes were determined by immunoblotting with the indicated antibodies. Representative confocal image ( E ) and quantification ( E , F ) of endogenous ciliary Smo after 48 h of siRNA-mediated depletion of mGprasp2 in PLCs. Note that levels of Alexa Fluor 488 (high and low) correlates with different Gprasp2 knock-down efficiency. Scale bar = 25 μm. > 100 cilia per condition were analyzed. All error bars indicate the mean ± SD of three independent experiments. Data were analyzed using a two tailed unpaired t -test (** = p
Figure Legend Snippet: Gprasp2 is required for ciliary localization of Smo and Shh target gene activation. ( A ) Shh-induced protein levels after 48h of siRNA-mediated knock-down of Gprasp2 in PLCs. ( B ) Mouse Gprasp2 siRNA target sequences with Multiple species ClustalW alignment to human and mouse Gprasp2 and indicated siRNA seed region position. Determination of Smo-Pifo complex formation after depletion of mGprasp2 ( C ) and rescue of mGprasp2 depletion by introducing hGPRASP2 ( D ). 48h post-transfection with the indicated siRNA duplexes or co-transfection with the indicated siRNA duplexes and HA-tagged hGPRASP2, PLCs were stimulated with Shh and the lysates were subsequently subjected to Strep-Tactin sepharose-coupled mPifo. Input (10%) and endogenous Smo protein complexes were determined by immunoblotting with the indicated antibodies. Representative confocal image ( E ) and quantification ( E , F ) of endogenous ciliary Smo after 48 h of siRNA-mediated depletion of mGprasp2 in PLCs. Note that levels of Alexa Fluor 488 (high and low) correlates with different Gprasp2 knock-down efficiency. Scale bar = 25 μm. > 100 cilia per condition were analyzed. All error bars indicate the mean ± SD of three independent experiments. Data were analyzed using a two tailed unpaired t -test (** = p

Techniques Used: Activation Assay, Transfection, Cotransfection, Two Tailed Test

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

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

Journal: Cell Reports

doi: 10.1016/j.celrep.2018.10.083

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

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

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

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

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

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

Journal: Cell Reports

doi: 10.1016/j.celrep.2018.10.083

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

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

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

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

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

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

Journal: Cell Reports

doi: 10.1016/j.celrep.2018.10.083

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

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

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

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

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

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

Journal: Cell Reports

doi: 10.1016/j.celrep.2018.10.083

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

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

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

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

60) Product Images from "TRUSS, a Novel Tumor Necrosis Factor Receptor 1 Scaffolding Protein That Mediates Activation of the Transcription Factor NF-?B"

Article Title: TRUSS, a Novel Tumor Necrosis Factor Receptor 1 Scaffolding Protein That Mediates Activation of the Transcription Factor NF-?B

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.22.8334-8344.2003

TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. (A) COS-7 cells were cotransfected with pFLAG.TNF-R1 and pHA.TRUSS, lysed, immunoprecipitated with hamster anti-TNF-R1 antagonist antibody, and analyzed by SDS-polyacrylamide gel electrophoresis. TRUSS was detected by Western blotting with anti-HA antibody. WCL, whole-cell lysate; IP, immunoprecipitation; NIgG, nonimmune rabbit IgG. (B) TRUSS interacts with TNF-R1 in U937 cells. Approximately 5 × 10 8 U937 cells were stimulated with human TNF-α for up to 10 min and lysed. Coimmunoprecipitating TRUSS and TRADD were detected by Western blotting. (C) Schematic representation of the GST-TNF-R1 cytoplasmic domain fusion proteins that were tested for their ability to bind TRUSS. MPR, membrane-proximal region; DD, death domain. (D) Interaction of TRUSS with the cytoplasmic domain of TNF-R1. COS-7 cells were transfected with 3 μg of pHA.TRUSS, lysed, and incubated with equal amounts of glutathione-Sepharose beads coated with the indicated GST-TNF-R1 cytoplasmic domain deletion mutants or GST alone. Coprecipitating proteins were detected by Western blotting with anti-HA antibody. (E) COS-7 cells were transfected with plasmids encoding either (i) FLAG-tagged full-length TNF-R1 (FLAG.TNF-R1 1-425), (ii) the FLAG-tagged TNF-R1 cytoplasmic domain (FLAG.TNF-R1 207-425), or (iii) the FLAG-tagged TNF-R1death domain (FLAG.TNF-R1 294-425). Lysates were incubated with equal amounts of GST-TRUSS- or GST-coated Sepharose beads, and the coprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with an anti-FLAG antibody to detect TNF-R1.
Figure Legend Snippet: TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. (A) COS-7 cells were cotransfected with pFLAG.TNF-R1 and pHA.TRUSS, lysed, immunoprecipitated with hamster anti-TNF-R1 antagonist antibody, and analyzed by SDS-polyacrylamide gel electrophoresis. TRUSS was detected by Western blotting with anti-HA antibody. WCL, whole-cell lysate; IP, immunoprecipitation; NIgG, nonimmune rabbit IgG. (B) TRUSS interacts with TNF-R1 in U937 cells. Approximately 5 × 10 8 U937 cells were stimulated with human TNF-α for up to 10 min and lysed. Coimmunoprecipitating TRUSS and TRADD were detected by Western blotting. (C) Schematic representation of the GST-TNF-R1 cytoplasmic domain fusion proteins that were tested for their ability to bind TRUSS. MPR, membrane-proximal region; DD, death domain. (D) Interaction of TRUSS with the cytoplasmic domain of TNF-R1. COS-7 cells were transfected with 3 μg of pHA.TRUSS, lysed, and incubated with equal amounts of glutathione-Sepharose beads coated with the indicated GST-TNF-R1 cytoplasmic domain deletion mutants or GST alone. Coprecipitating proteins were detected by Western blotting with anti-HA antibody. (E) COS-7 cells were transfected with plasmids encoding either (i) FLAG-tagged full-length TNF-R1 (FLAG.TNF-R1 1-425), (ii) the FLAG-tagged TNF-R1 cytoplasmic domain (FLAG.TNF-R1 207-425), or (iii) the FLAG-tagged TNF-R1death domain (FLAG.TNF-R1 294-425). Lysates were incubated with equal amounts of GST-TRUSS- or GST-coated Sepharose beads, and the coprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with an anti-FLAG antibody to detect TNF-R1.

Techniques Used: Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Transfection, Incubation

TRUSS interacts with TRADD, TRAF2, RIP, IKKα, IKKβ, and IKKγ. (A) Interaction of TRUSS with TRADD, TRAF2, and components of the IKK complex. Results of GST- and His-tagged pull-down assays are shown in the left column. COS-7 cells were transfected with plasmids encoding Myc-tagged TRADD, FLAG-tagged TRAF2, FLAG-tagged IKK-α, or Myc-tagged IKK-β, lysed, and incubated with GST-TRUSS or GST-coated Sepharose beads. Interactions between IKK-γ and TRUSS were detected by incubating recombinant His-tagged IKK-γ with lysates from HA-tagged-TRUSS-transfected COS-7 cells. The complexes were captured by binding to Ni-agarose beads. All coprecipitating proteins were detected by SDS-polyacrylamide gel electrophoresis and Western blotting with the antibodies indicated. Results of coimmunoprecipitation assays are shown in the right column. COS-7 cells were cotransfected with plasmids encoding Myc-tagged TRADD, FLAG-tagged TRAF2, IKK-α, IKK-β-,or IKK-γ together with HA-tagged TRUSS, lysed, and immunoprecipitated with anti-HA antibody (or anti-IKK-γ). Coimmunoprecipitating proteins were detected by immunoblotting with the relevant antibody as indicated. WCL, whole-cell lysate; IP, immunoprecipitation; NIgG, nonimmune rabbit IgG; NTA, nitrilotriacetic acid. (B) RIP is present in a complex with TRUSS. COS-7 cells were cotransfected with HA.TRUSS and FLAG-tagged RIP, lysed, and immunoprecipitated with anti-HA antibody or anti-FLAG antibody. Coimmunoprecipitating proteins were detected by Western blotting with anti-HA (TRUSS) or anti-FLAG (RIP) antibodies. (C) TRUSS coimmunoprecipitates with endogenous TRADD, TRAF-2, and IKK in HEK293 cells. HEK293 cells were transfected with HA-tagged TRUSS (top) or empty vector (bottom), lysed, and immunoprecipitated with anti-TNF-R1, anti-TRADD, anti-TRAF2, anti-IKK-α, anti-RIP, or with anti-DR3 antibody as a negative control. Coprecipitating TRUSS was detected by Western blotting.
Figure Legend Snippet: TRUSS interacts with TRADD, TRAF2, RIP, IKKα, IKKβ, and IKKγ. (A) Interaction of TRUSS with TRADD, TRAF2, and components of the IKK complex. Results of GST- and His-tagged pull-down assays are shown in the left column. COS-7 cells were transfected with plasmids encoding Myc-tagged TRADD, FLAG-tagged TRAF2, FLAG-tagged IKK-α, or Myc-tagged IKK-β, lysed, and incubated with GST-TRUSS or GST-coated Sepharose beads. Interactions between IKK-γ and TRUSS were detected by incubating recombinant His-tagged IKK-γ with lysates from HA-tagged-TRUSS-transfected COS-7 cells. The complexes were captured by binding to Ni-agarose beads. All coprecipitating proteins were detected by SDS-polyacrylamide gel electrophoresis and Western blotting with the antibodies indicated. Results of coimmunoprecipitation assays are shown in the right column. COS-7 cells were cotransfected with plasmids encoding Myc-tagged TRADD, FLAG-tagged TRAF2, IKK-α, IKK-β-,or IKK-γ together with HA-tagged TRUSS, lysed, and immunoprecipitated with anti-HA antibody (or anti-IKK-γ). Coimmunoprecipitating proteins were detected by immunoblotting with the relevant antibody as indicated. WCL, whole-cell lysate; IP, immunoprecipitation; NIgG, nonimmune rabbit IgG; NTA, nitrilotriacetic acid. (B) RIP is present in a complex with TRUSS. COS-7 cells were cotransfected with HA.TRUSS and FLAG-tagged RIP, lysed, and immunoprecipitated with anti-HA antibody or anti-FLAG antibody. Coimmunoprecipitating proteins were detected by Western blotting with anti-HA (TRUSS) or anti-FLAG (RIP) antibodies. (C) TRUSS coimmunoprecipitates with endogenous TRADD, TRAF-2, and IKK in HEK293 cells. HEK293 cells were transfected with HA-tagged TRUSS (top) or empty vector (bottom), lysed, and immunoprecipitated with anti-TNF-R1, anti-TRADD, anti-TRAF2, anti-IKK-α, anti-RIP, or with anti-DR3 antibody as a negative control. Coprecipitating TRUSS was detected by Western blotting.

Techniques Used: Transfection, Incubation, Recombinant, Binding Assay, Polyacrylamide Gel Electrophoresis, Western Blot, Immunoprecipitation, Plasmid Preparation, Negative Control

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

Article Title: Trs20 is Required for TRAPP II Assembly

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/tra.12065

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

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

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

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

62) Product Images from "Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets"

Article Title: Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E15-04-0247

CREB-HΔTMC phosphorylation by CKII and GSK-3 on distinct motifs within and adjacent to the DSG region, respectively. (a) Equal amounts of purified GST, GST-wt, or GST-DSG protein bound to glutathione-agarose beads were incubated with purified GSK-3b or CKII and analyzed by SDS–PAGE and autoradiography. The amount of proteins in the reaction is shown by total protein staining (CBB), and the phosphorylated species is shown for each kinase. The GST control migrates at a lower position but has been transposed for ease of direct comparison. (b) Replicate equivalent samples of the GST-wt protein were incubated in vitro without or with a primary kinase as indicated (1st) and the beads washed to remove the kinase and exchange buffers and then incubated without or with a second kinase (2nd). The different combinations and order are indicated. Samples were then analyzed by SDS–PAGE and autoradiography. (c) Equal amounts of the GST-wt (lanes 1, 3, 5, and 7) and GST-DSG mutant (lanes 2, 4, 6, and 8), as in a, were incubated with the various combinations of primary and secondary kinases as indicated. (d) Equal amounts of purified GST, GST-3Sd, GST-DSG, or GST-3Sd+DSG protein were incubated with purified GSK-3 or CKII and subsequently analyzed by SDS–PAGE and autoradiography. The equalized amount of the proteins in the reaction is shown by total protein staining (CBB), and the phosphorylated species is shown for each kinase and mutant as indicated.
Figure Legend Snippet: CREB-HΔTMC phosphorylation by CKII and GSK-3 on distinct motifs within and adjacent to the DSG region, respectively. (a) Equal amounts of purified GST, GST-wt, or GST-DSG protein bound to glutathione-agarose beads were incubated with purified GSK-3b or CKII and analyzed by SDS–PAGE and autoradiography. The amount of proteins in the reaction is shown by total protein staining (CBB), and the phosphorylated species is shown for each kinase. The GST control migrates at a lower position but has been transposed for ease of direct comparison. (b) Replicate equivalent samples of the GST-wt protein were incubated in vitro without or with a primary kinase as indicated (1st) and the beads washed to remove the kinase and exchange buffers and then incubated without or with a second kinase (2nd). The different combinations and order are indicated. Samples were then analyzed by SDS–PAGE and autoradiography. (c) Equal amounts of the GST-wt (lanes 1, 3, 5, and 7) and GST-DSG mutant (lanes 2, 4, 6, and 8), as in a, were incubated with the various combinations of primary and secondary kinases as indicated. (d) Equal amounts of purified GST, GST-3Sd, GST-DSG, or GST-3Sd+DSG protein were incubated with purified GSK-3 or CKII and subsequently analyzed by SDS–PAGE and autoradiography. The equalized amount of the proteins in the reaction is shown by total protein staining (CBB), and the phosphorylated species is shown for each kinase and mutant as indicated.

Techniques Used: Purification, Incubation, SDS Page, Autoradiography, Staining, In Vitro, Mutagenesis

Direct phosphorylation-dependent interaction between CREB-H and Fbw1a. Cells were transfected with Flag-tagged Fbw1a and soluble extracts made. Equal amounts of purified GST, GST-wt, or GST-DSG protein bound to glutathione-agarose beads were incubated without (–) or with (+) purified GSK-3 and CKII for 30 min before the addition of the Fbw1a- transfected soluble extracts. After further incubation, the beads were isolated, washed extensively, and then analyzed by SDS–PAGE and Western blotting for Fbw1a and for GST fusion protein in the pull-down (Fbw1a and GST panels). The input sample (Inp) represents 1/50 of the total input, and 1/5 of the pull-down material was analyzed in each case. Equal amounts of total GST-fusion proteins were incubated in each case and present in the pull-down as detected by anti-GST antibody (bottom GST panel). Specific interaction with wt was observed but only after phosphorylation by GSK-3 and CKII. In parallel, no significant interaction was observed for the GST control or for GST-DSG.
Figure Legend Snippet: Direct phosphorylation-dependent interaction between CREB-H and Fbw1a. Cells were transfected with Flag-tagged Fbw1a and soluble extracts made. Equal amounts of purified GST, GST-wt, or GST-DSG protein bound to glutathione-agarose beads were incubated without (–) or with (+) purified GSK-3 and CKII for 30 min before the addition of the Fbw1a- transfected soluble extracts. After further incubation, the beads were isolated, washed extensively, and then analyzed by SDS–PAGE and Western blotting for Fbw1a and for GST fusion protein in the pull-down (Fbw1a and GST panels). The input sample (Inp) represents 1/50 of the total input, and 1/5 of the pull-down material was analyzed in each case. Equal amounts of total GST-fusion proteins were incubated in each case and present in the pull-down as detected by anti-GST antibody (bottom GST panel). Specific interaction with wt was observed but only after phosphorylation by GSK-3 and CKII. In parallel, no significant interaction was observed for the GST control or for GST-DSG.

Techniques Used: Transfection, Purification, Incubation, Isolation, SDS Page, Western Blot

63) Product Images from "Molecular clearance of ataxin-3 is regulated by a mammalian E4"

Article Title: Molecular clearance of ataxin-3 is regulated by a mammalian E4

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600081

Interaction between VCP and ataxin-3 in vivo and in vitro . ( A ) HEK293T cells were transiently transfected with an expression vector for Myc–ataxin-3(79Q) or with the empty vector as a control. Cell lysates were subjected to immunoprecipitation with anti-Myc, and the resulting precipitates as well as the original lysates (load) were subjected to immunoblot analysis with anti-VCP or anti-Myc. ( B ) GST, GST–ataxin-3(0Q, 13Q, or 79Q), or GST-fusion proteins of the indicated ataxin-3 deletion mutants were incubated for 30 min at room temperature with recombinant HA–VCP. GST or the GST-fusion proteins were then precipitated with glutathione–sepharose beads and subjected to immunoblot analysis with anti-HA (top panel) or anti-GST (middle panel). A portion (10%) of the input binding mixture was also subjected directly to immunoblot analysis with anti-HA (bottom panel). ( C ) Schematic representation of the ataxin-3 deletion mutants and summary of the data obtained from the in vitro assay of the binding of ataxin-3 derivatives to VCP.
Figure Legend Snippet: Interaction between VCP and ataxin-3 in vivo and in vitro . ( A ) HEK293T cells were transiently transfected with an expression vector for Myc–ataxin-3(79Q) or with the empty vector as a control. Cell lysates were subjected to immunoprecipitation with anti-Myc, and the resulting precipitates as well as the original lysates (load) were subjected to immunoblot analysis with anti-VCP or anti-Myc. ( B ) GST, GST–ataxin-3(0Q, 13Q, or 79Q), or GST-fusion proteins of the indicated ataxin-3 deletion mutants were incubated for 30 min at room temperature with recombinant HA–VCP. GST or the GST-fusion proteins were then precipitated with glutathione–sepharose beads and subjected to immunoblot analysis with anti-HA (top panel) or anti-GST (middle panel). A portion (10%) of the input binding mixture was also subjected directly to immunoblot analysis with anti-HA (bottom panel). ( C ) Schematic representation of the ataxin-3 deletion mutants and summary of the data obtained from the in vitro assay of the binding of ataxin-3 derivatives to VCP.

Techniques Used: In Vivo, In Vitro, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Incubation, Recombinant, Binding Assay

Interaction of E4B with VCP and ataxin-3. ( A ) HEK293T cells transiently expressing HA–VCP and FLAG–E4B were subjected to immunoprecipitation with anti-FLAG, and the resulting precipitates were subjected to immunoblot analysis with anti-HA or anti-FLAG. A portion (10%) of the input lysates was also subjected directly to immunoblot analysis with anti-HA and anti-FLAG. ( B ) Recombinant GST or GST–VCP was mixed with recombinant FLAG-tagged wild-type (Wt) E4B, a deletion mutant lacking the U-box domain (ΔU), or an NH 2 -terminal deletion mutant (ΔN). Proteins precipitated with glutathione–sepharose beads were then subjected to immunoblot analysis with anti-FLAG (top panel) or anti-GST (middle panels). A portion (10%) of the input binding mixture was also subjected directly to immunoblot analysis with anti-FLAG (bottom panel). ( C ) An HMW fraction (Superose 6, fraction 13) of rabbit reticulocyte lysate was subjected to immunoprecipitation with anti-E4B or normal immunoglobulin, and the resulting precipitates were subjected to immunoblot analysis with anti-VCP and anti-E4B. ( D ) GST or GST–ataxin-3(79Q) was mixed with FLAG–E4B and HA–VCP, as indicated, and then precipitated with glutathione–sepharose beads. The precipitated proteins were subjected to immunoblot analysis with anti-FLAG, anti-HA, or anti-GST.
Figure Legend Snippet: Interaction of E4B with VCP and ataxin-3. ( A ) HEK293T cells transiently expressing HA–VCP and FLAG–E4B were subjected to immunoprecipitation with anti-FLAG, and the resulting precipitates were subjected to immunoblot analysis with anti-HA or anti-FLAG. A portion (10%) of the input lysates was also subjected directly to immunoblot analysis with anti-HA and anti-FLAG. ( B ) Recombinant GST or GST–VCP was mixed with recombinant FLAG-tagged wild-type (Wt) E4B, a deletion mutant lacking the U-box domain (ΔU), or an NH 2 -terminal deletion mutant (ΔN). Proteins precipitated with glutathione–sepharose beads were then subjected to immunoblot analysis with anti-FLAG (top panel) or anti-GST (middle panels). A portion (10%) of the input binding mixture was also subjected directly to immunoblot analysis with anti-FLAG (bottom panel). ( C ) An HMW fraction (Superose 6, fraction 13) of rabbit reticulocyte lysate was subjected to immunoprecipitation with anti-E4B or normal immunoglobulin, and the resulting precipitates were subjected to immunoblot analysis with anti-VCP and anti-E4B. ( D ) GST or GST–ataxin-3(79Q) was mixed with FLAG–E4B and HA–VCP, as indicated, and then precipitated with glutathione–sepharose beads. The precipitated proteins were subjected to immunoblot analysis with anti-FLAG, anti-HA, or anti-GST.

Techniques Used: Expressing, Immunoprecipitation, Recombinant, Mutagenesis, Binding Assay

64) Product Images from "Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase"

Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007282

CLCuMuV C4 protein interacts with NbSAMS2 in vitro and in vivo . (A) CLCuMuV C4 protein co-immunoprecipitated with NbSAMS2. Total protein extracts were immunoprecipitated with anti-GFP beads and then monitored by immunoblotting (IB) using anti-GFP or anti-HA antibodies. cLUC represents c-terminal fragment of the firefly luciferase. (B) GST pull-down assay showed the interaction between C4 and NbSAMS2. Total soluble proteins of E . coli expressing GST-NbSAMS2 or GST were incubated with C4- His or C4 R13A -His immobilized on glutathione-sepharose beads and monitored by anti-His antibody. (C) BiFC assay showed the interaction between C4 and NbSAMS2. Cells were photographed 60 hpi using confocal laser scanning microscope. Bar scale represents 50 μm. (D) Western blot analyses of BiFC construct combinations from the same experiments as in (C). All combinations were detected with anti-GFP polyclonal antibody.
Figure Legend Snippet: CLCuMuV C4 protein interacts with NbSAMS2 in vitro and in vivo . (A) CLCuMuV C4 protein co-immunoprecipitated with NbSAMS2. Total protein extracts were immunoprecipitated with anti-GFP beads and then monitored by immunoblotting (IB) using anti-GFP or anti-HA antibodies. cLUC represents c-terminal fragment of the firefly luciferase. (B) GST pull-down assay showed the interaction between C4 and NbSAMS2. Total soluble proteins of E . coli expressing GST-NbSAMS2 or GST were incubated with C4- His or C4 R13A -His immobilized on glutathione-sepharose beads and monitored by anti-His antibody. (C) BiFC assay showed the interaction between C4 and NbSAMS2. Cells were photographed 60 hpi using confocal laser scanning microscope. Bar scale represents 50 μm. (D) Western blot analyses of BiFC construct combinations from the same experiments as in (C). All combinations were detected with anti-GFP polyclonal antibody.

Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Luciferase, Pull Down Assay, Expressing, Incubation, Bimolecular Fluorescence Complementation Assay, Laser-Scanning Microscopy, Western Blot, Construct

Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p
Figure Legend Snippet: Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

Techniques Used: Infection, Plasmid Preparation, Southern Blot, Labeling, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

65) Product Images from "Molecular basis of AKAP79 regulation by calmodulin"

Article Title: Molecular basis of AKAP79 regulation by calmodulin

Journal: Nature Communications

doi: 10.1038/s41467-017-01715-w

Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM sepharose. Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P
Figure Legend Snippet: Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM sepharose. Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P

Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Derivative Assay, Purification, Variant Assay, Incubation

Delineation of key residues in AKAP79 required for CaM binding. a Pull-down of either WT, Δ33–48, Δ79–86, or Δ391–400 FLAG-tagged-AKAP79 (inputs shown in bottom panel) with either CaM sepharose (top panel) or cAMP agarose (middle panel). AKAP79 was detected by anti-FLAG immunoblotting. The experiment was performed in triplicate with each replicate producing the same pattern of bands. b Sequence LOGO for AKAP5 gene products aligned with predicted helical region. The cross-linking cluster between AKAP79 positions 90–99 and K94 in CaM is indicated along with the boundaries of peptides used in the following panels. c – e Determination of inhibitory constants for the peptides outlined in ( b ) in disrupting interaction between biotin-CaM and GST-AKAP79 (1–153) detected using the alphascreen assay ( n = 4 for all data points). K i constants were determined for the 9-mer and 11-mer peptides c , 16-mer and 20-mer peptides d , and for either WT or L101A 26-mer peptides e .***P
Figure Legend Snippet: Delineation of key residues in AKAP79 required for CaM binding. a Pull-down of either WT, Δ33–48, Δ79–86, or Δ391–400 FLAG-tagged-AKAP79 (inputs shown in bottom panel) with either CaM sepharose (top panel) or cAMP agarose (middle panel). AKAP79 was detected by anti-FLAG immunoblotting. The experiment was performed in triplicate with each replicate producing the same pattern of bands. b Sequence LOGO for AKAP5 gene products aligned with predicted helical region. The cross-linking cluster between AKAP79 positions 90–99 and K94 in CaM is indicated along with the boundaries of peptides used in the following panels. c – e Determination of inhibitory constants for the peptides outlined in ( b ) in disrupting interaction between biotin-CaM and GST-AKAP79 (1–153) detected using the alphascreen assay ( n = 4 for all data points). K i constants were determined for the 9-mer and 11-mer peptides c , 16-mer and 20-mer peptides d , and for either WT or L101A 26-mer peptides e .***P

Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Sequencing, Amplified Luminescent Proximity Homogenous Assay

66) Product Images from "Nuclear Import of UBL-Domain Protein Mdy2 Is Required for Heat-Induced Stress Response in Saccharomyces cerevisiae"

Article Title: Nuclear Import of UBL-Domain Protein Mdy2 Is Required for Heat-Induced Stress Response in Saccharomyces cerevisiae

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052956

Binding assays of Get4 and Sgt2 with Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins. (A) HZH686 (W303-1A mdy2 Δ ) cells were transformed with CEN expression vectors encoding Myc, Myc-tagged Mdy2 (Myc-Mdy2), Myc-Mdy2-NLS, Myc-Mdy2-NES, GST, and GST-tagged Get4 (GST-Get4) under the control of GAL1 promoter. Cells were grown in SRG to log phase (see Material and Methods), whole cell extracts were prepared and GST-Get4 was precipitated using Glutathione -Sepharose 4B. The pulldown was then tested for the presence of Mdy2 association by probing a Western blot with anti-Myc Ab (top panel). To monitor pulldown recovery, the level of GST-Mdy2 in the binding assay was measured by probing the same membrane with anti-GST antibody (middle panel). Expression levels of Myc-Mdy2 in the whole cell extracts used for binding assay were measured on a Western blot (bottom panel). (B) HZH686 (W303-1A mdy2 Δ ) cells were transformed with expression vectors encoding Myc-tagged Mdy2 variants as in (A) and GST-tagged Sgt2 (GST-Sgt2) under the control of GAL1 promoter. Cells were grown in SRG to log phase, whole cell extracts were prepared, and GST-Sgt2 was precipitated using Glutathione -Sepharose 4B. The presence of Mdy2 in the pulldown was confirmed by probing a Western blot with anti-Myc antibody (top panel). To monitor binding recovery the level of GST-Sgt2 in the pulldown was measured by probing the same membrane with anti-GST Ab (middle panel). Expression levels of Myc-Mdy2 in the whole cell extracts used for pulldown were measured on Western blots (bottom panel).
Figure Legend Snippet: Binding assays of Get4 and Sgt2 with Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins. (A) HZH686 (W303-1A mdy2 Δ ) cells were transformed with CEN expression vectors encoding Myc, Myc-tagged Mdy2 (Myc-Mdy2), Myc-Mdy2-NLS, Myc-Mdy2-NES, GST, and GST-tagged Get4 (GST-Get4) under the control of GAL1 promoter. Cells were grown in SRG to log phase (see Material and Methods), whole cell extracts were prepared and GST-Get4 was precipitated using Glutathione -Sepharose 4B. The pulldown was then tested for the presence of Mdy2 association by probing a Western blot with anti-Myc Ab (top panel). To monitor pulldown recovery, the level of GST-Mdy2 in the binding assay was measured by probing the same membrane with anti-GST antibody (middle panel). Expression levels of Myc-Mdy2 in the whole cell extracts used for binding assay were measured on a Western blot (bottom panel). (B) HZH686 (W303-1A mdy2 Δ ) cells were transformed with expression vectors encoding Myc-tagged Mdy2 variants as in (A) and GST-tagged Sgt2 (GST-Sgt2) under the control of GAL1 promoter. Cells were grown in SRG to log phase, whole cell extracts were prepared, and GST-Sgt2 was precipitated using Glutathione -Sepharose 4B. The presence of Mdy2 in the pulldown was confirmed by probing a Western blot with anti-Myc antibody (top panel). To monitor binding recovery the level of GST-Sgt2 in the pulldown was measured by probing the same membrane with anti-GST Ab (middle panel). Expression levels of Myc-Mdy2 in the whole cell extracts used for pulldown were measured on Western blots (bottom panel).

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

Mdy2 co-localize and interact with Pab1. (A) Mdy2 co-localize with Pab1 following heat stress and treatment with sodium azide. GFP-Mdy2 and Pab1-RFP was visualized by fluorescence microscopy in a mdy2 Δ strain transformed with plasmids containing GFP-Mdy2 (upper row), GFP-Mdy2-ΔNLS (middle row) or GFP-Mdy2-ΔNES (lower panel), and Pab1-RFP, after a temperature shift to 46°C (left panel) and after treatment with sodium azide (NaN 3 ) (right panel). In the overlay pictures (merge), overlap of the colors appears yellow. GFP-Mdy2 and GFP-Mdy2-ΔNES but not GFP-Mdy2-ΔNLS are predominantly nuclear in control (Con) conditions at 28°C (right panel). (B) Mdy2 interacts with Pab1. Cell lysates from the GST-tagged Mdy2 strains were precipitated (P) with Glutathione Sepharose 4B. Following washing, the resin was eluted with glutathione. Eluted proteins were resolved by SDS-PAGE and visualized by immunobloting (control, IB) and Coomassie blue staining (Coomassie). Protein identities were established by mass spectrometry analysis. (C) Extracts from yeast strains HZH686 (W303-1A mdy2 Δ ) coexpressing GST alone (GST) or GST-tagged Pab1 (GST-Pab1) with Myc alone (Myc), Myc-tagged Mdy2 (Myc-Mdy2), Myc-tagged Mdy2-ΔNLS (Myc-Mdy2-ΔNLS) or Myc-tagged Mdy2-ΔNES (Myc-Mdy2-ΔNES) were subjected to pulldown using Glutathione Sepharose 4B as in Figure 4 . The coprecipitation of indicated Myc-tagged Mdy2 proteins in the pulldown was confirmed by probing a Western blot with anti-Myc Ab (top panel, Co-P: Myc). To monitor pulldown recovery, the level of GST-Pab1 in the pulldown was measured by probing the same membrane with anti-GST Ab (second panel from the top, P: GST). Expression levels of indicated Myc-tagged Mdy2 proteins and GST-Pab1 in whole cell extracts (Extract) used for pulldown were measured on Western blots (third and fourth panels from top, IB:Myc and IB:GST, respectively).
Figure Legend Snippet: Mdy2 co-localize and interact with Pab1. (A) Mdy2 co-localize with Pab1 following heat stress and treatment with sodium azide. GFP-Mdy2 and Pab1-RFP was visualized by fluorescence microscopy in a mdy2 Δ strain transformed with plasmids containing GFP-Mdy2 (upper row), GFP-Mdy2-ΔNLS (middle row) or GFP-Mdy2-ΔNES (lower panel), and Pab1-RFP, after a temperature shift to 46°C (left panel) and after treatment with sodium azide (NaN 3 ) (right panel). In the overlay pictures (merge), overlap of the colors appears yellow. GFP-Mdy2 and GFP-Mdy2-ΔNES but not GFP-Mdy2-ΔNLS are predominantly nuclear in control (Con) conditions at 28°C (right panel). (B) Mdy2 interacts with Pab1. Cell lysates from the GST-tagged Mdy2 strains were precipitated (P) with Glutathione Sepharose 4B. Following washing, the resin was eluted with glutathione. Eluted proteins were resolved by SDS-PAGE and visualized by immunobloting (control, IB) and Coomassie blue staining (Coomassie). Protein identities were established by mass spectrometry analysis. (C) Extracts from yeast strains HZH686 (W303-1A mdy2 Δ ) coexpressing GST alone (GST) or GST-tagged Pab1 (GST-Pab1) with Myc alone (Myc), Myc-tagged Mdy2 (Myc-Mdy2), Myc-tagged Mdy2-ΔNLS (Myc-Mdy2-ΔNLS) or Myc-tagged Mdy2-ΔNES (Myc-Mdy2-ΔNES) were subjected to pulldown using Glutathione Sepharose 4B as in Figure 4 . The coprecipitation of indicated Myc-tagged Mdy2 proteins in the pulldown was confirmed by probing a Western blot with anti-Myc Ab (top panel, Co-P: Myc). To monitor pulldown recovery, the level of GST-Pab1 in the pulldown was measured by probing the same membrane with anti-GST Ab (second panel from the top, P: GST). Expression levels of indicated Myc-tagged Mdy2 proteins and GST-Pab1 in whole cell extracts (Extract) used for pulldown were measured on Western blots (third and fourth panels from top, IB:Myc and IB:GST, respectively).

Techniques Used: Fluorescence, Microscopy, Transformation Assay, SDS Page, Western Blot, Staining, Mass Spectrometry, Expressing

67) Product Images from "Ubiquitination of Lysine 867 of the Human SETDB1 Protein Upregulates Its Histone H3 Lysine 9 (H3K9) Methyltransferase Activity"

Article Title: Ubiquitination of Lysine 867 of the Human SETDB1 Protein Upregulates Its Histone H3 Lysine 9 (H3K9) Methyltransferase Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0165766

The PTMs of SETDB1 are associated with its H3K9 methyltransferase activity in HeLa cells. (A) Schematic representation of the domain structure of human SETDB1 and the deletion mutants. Amino acid sequence is numbered in accordance with the UniProt numbering scheme; the tandem Tudor domains, methyl-CpG-binding domain (MBD), pre-SET domain, SET domain, and post-SET domain are indicated. (B) SETDB1 proteins were expressed as GST fusion protein in HeLa cells and purified on glutathione-sepharose beads. The purified SETDB1 proteins were resolved on 5% SDS-PAGE, and electroblotted onto PVDF membrane. Western blot analysis of GST-SETDB1 proteins was probed with anti-GST antibody. (C) H3K9 methyltransferase activity of GST affinity-purified SETDB1 proteins in HeLa cells was measured. The values represent means±SEM (n = 3).
Figure Legend Snippet: The PTMs of SETDB1 are associated with its H3K9 methyltransferase activity in HeLa cells. (A) Schematic representation of the domain structure of human SETDB1 and the deletion mutants. Amino acid sequence is numbered in accordance with the UniProt numbering scheme; the tandem Tudor domains, methyl-CpG-binding domain (MBD), pre-SET domain, SET domain, and post-SET domain are indicated. (B) SETDB1 proteins were expressed as GST fusion protein in HeLa cells and purified on glutathione-sepharose beads. The purified SETDB1 proteins were resolved on 5% SDS-PAGE, and electroblotted onto PVDF membrane. Western blot analysis of GST-SETDB1 proteins was probed with anti-GST antibody. (C) H3K9 methyltransferase activity of GST affinity-purified SETDB1 proteins in HeLa cells was measured. The values represent means±SEM (n = 3).

Techniques Used: Activity Assay, Sequencing, Binding Assay, Purification, SDS Page, Western Blot, Affinity Purification

Ubiquitination of K867 of GST-SETDB1 (570–1291) upregulates its H3K9 methyltransferase activity in HeLa cells. (A) SETDB1 proteins were expressed as GST fusion proteins in HeLa cells and purified on glutathione-sepharose beads. The purified SETDB1 proteins were resolved on 5% SDS-PAGE, and electroblotted onto PVDF membranes. Western blot analyses of GST-SETDB1 proteins were probed with anti-SETDB1 antibody or anti-ubiquitin antibody. (B) H3K9 methyltransferase activity of the GST affinity-purified SETDB1 proteins in HeLa cells was measured. The values represent means±SEM (n = 3). ** P
Figure Legend Snippet: Ubiquitination of K867 of GST-SETDB1 (570–1291) upregulates its H3K9 methyltransferase activity in HeLa cells. (A) SETDB1 proteins were expressed as GST fusion proteins in HeLa cells and purified on glutathione-sepharose beads. The purified SETDB1 proteins were resolved on 5% SDS-PAGE, and electroblotted onto PVDF membranes. Western blot analyses of GST-SETDB1 proteins were probed with anti-SETDB1 antibody or anti-ubiquitin antibody. (B) H3K9 methyltransferase activity of the GST affinity-purified SETDB1 proteins in HeLa cells was measured. The values represent means±SEM (n = 3). ** P

Techniques Used: Activity Assay, Purification, SDS Page, Western Blot, Affinity Purification

68) Product Images from "Structural Basis for the Function of the Saccharomyces cerevisiae Gfd1 Protein in mRNA Nuclear Export *"

Article Title: Structural Basis for the Function of the Saccharomyces cerevisiae Gfd1 Protein in mRNA Nuclear Export *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.107276

nab2-Y34A shows impaired binding to Gfd1 and interacts genetically with rat8-2 (dbp5 ). A , the Nab2-N mutant nab2-Y34A does not interact with Gfd1 in vitro , but Nab2-N mutants L55W and F56D retain their ability to bind Gfd1 in vitro . Recombinant GST, GST-Nab2-N-wild-type (WT), or GST-Nab2-N mutant (Y34A, L55W, or F56D) was incubated with recombinant His-tagged Gfd1 (His-Gfd1) and glutathione-Sepharose beads as described under “Experimental Procedures.” Bound fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Unbound fractions were analyzed by immunoblotting with anti-His antibody. B , nab2-Y34A cells are viable and grow similarly to wild-type NAB2 cells. nab2 Δ cells maintained by a NAB2 URA3 plasmid and containing vector alone, NAB2 , nab2 -Δ N , nab2-Y34A , nab2-L55W , or nab2-F56D LEU2 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on control and 5-fluoroorotic acid ( 5-FOA ) plates. Cells were grown at 25, 30, and 37 °C. C , rat8-2 (dbp5 ) nab2-Y34A cells have a slow growth phenotype. nab2 Δ rat8-2 (dbp5 ) cells containing NAB2 , nab2-Y34A , nab2-L55W , or nab2-F56D LEU2 test plasmids as the sole copy of NAB2 were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Leu − minimal medium plates. Cells were grown at 25, 30, and, 32 °C. D , growth curve analysis of rat8-2 (dbp5) nab2-Y34A cells confirms that they grow more slowly than rat8-2 (dbp5) NAB2 cells. rat8-2 (dbp5 ) nab2 Δ cells carrying NAB2 , nab2-Y34A , nab2-L55W , or nab2-F56D plasmids as the sole copy of Nab2 were grown to saturation and diluted, and their optical density ( OD ) was measured at A 600 for 46 h as described under “Experimental Procedures.” E , nab2 mutants are expressed at similar levels. To examine the protein level of each nab2 variant, rat8-2 (dbp5 ) cells containing NAB2 or nab2 variant plasmids were grown at 30 °C, and whole cell lysates prepared from these cells were analyzed by immunoblotting with a polyclonal anti-Nab2 antibody. As a loading control, Pgk1 protein levels in each lysate were detected with a monoclonal anti-Pgk1 antibody. The nab2-Y34A protein level was comparable with that seen with wild-type and, although the levels of the two controls, nab2-L55W and nab2-F56D, were a little lower, this did not produce any phenotype. F , overexpression of GFD1 suppresses the slow growth phenotype of rat8-2 (dbp5) nab2-Y34A cells. nab2 Δ rat8-2 (dbp5 ) cells containing NAB2 or nab2-Y34A LEU2 plasmids as the sole copy of NAB2 and vector alone or GFD1 TRP1 plasmid were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Leu − Trp − minimal medium plates. Cells were grown at 25, 30, and, 32 °C.
Figure Legend Snippet: nab2-Y34A shows impaired binding to Gfd1 and interacts genetically with rat8-2 (dbp5 ). A , the Nab2-N mutant nab2-Y34A does not interact with Gfd1 in vitro , but Nab2-N mutants L55W and F56D retain their ability to bind Gfd1 in vitro . Recombinant GST, GST-Nab2-N-wild-type (WT), or GST-Nab2-N mutant (Y34A, L55W, or F56D) was incubated with recombinant His-tagged Gfd1 (His-Gfd1) and glutathione-Sepharose beads as described under “Experimental Procedures.” Bound fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Unbound fractions were analyzed by immunoblotting with anti-His antibody. B , nab2-Y34A cells are viable and grow similarly to wild-type NAB2 cells. nab2 Δ cells maintained by a NAB2 URA3 plasmid and containing vector alone, NAB2 , nab2 -Δ N , nab2-Y34A , nab2-L55W , or nab2-F56D LEU2 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on control and 5-fluoroorotic acid ( 5-FOA ) plates. Cells were grown at 25, 30, and 37 °C. C , rat8-2 (dbp5 ) nab2-Y34A cells have a slow growth phenotype. nab2 Δ rat8-2 (dbp5 ) cells containing NAB2 , nab2-Y34A , nab2-L55W , or nab2-F56D LEU2 test plasmids as the sole copy of NAB2 were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Leu − minimal medium plates. Cells were grown at 25, 30, and, 32 °C. D , growth curve analysis of rat8-2 (dbp5) nab2-Y34A cells confirms that they grow more slowly than rat8-2 (dbp5) NAB2 cells. rat8-2 (dbp5 ) nab2 Δ cells carrying NAB2 , nab2-Y34A , nab2-L55W , or nab2-F56D plasmids as the sole copy of Nab2 were grown to saturation and diluted, and their optical density ( OD ) was measured at A 600 for 46 h as described under “Experimental Procedures.” E , nab2 mutants are expressed at similar levels. To examine the protein level of each nab2 variant, rat8-2 (dbp5 ) cells containing NAB2 or nab2 variant plasmids were grown at 30 °C, and whole cell lysates prepared from these cells were analyzed by immunoblotting with a polyclonal anti-Nab2 antibody. As a loading control, Pgk1 protein levels in each lysate were detected with a monoclonal anti-Pgk1 antibody. The nab2-Y34A protein level was comparable with that seen with wild-type and, although the levels of the two controls, nab2-L55W and nab2-F56D, were a little lower, this did not produce any phenotype. F , overexpression of GFD1 suppresses the slow growth phenotype of rat8-2 (dbp5) nab2-Y34A cells. nab2 Δ rat8-2 (dbp5 ) cells containing NAB2 or nab2-Y34A LEU2 plasmids as the sole copy of NAB2 and vector alone or GFD1 TRP1 plasmid were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Leu − Trp − minimal medium plates. Cells were grown at 25, 30, and, 32 °C.

Techniques Used: Binding Assay, Mutagenesis, In Vitro, Recombinant, Incubation, SDS Page, Staining, Plasmid Preparation, Variant Assay, Over Expression

gfd1 mutants that are impaired for binding to Nab2 show only partial suppression of rat8-2 (dbp5 ) temperature-sensitive growth. A , gfd1 mutants with alanine insertions ( gfd1-Lys 135 -A-Lys 136 ; gfd1-Lys 135 -AA-Lys 136 ) or deletions ( gfd1 -Δ 122–143 ; gfd -Δ 130–143 ) in the Nab2-binding α-helix only partially suppress the temperature-sensitive growth of rat8-2 (dbp5 ) cells. rat8-2 (dbp5 ) cells containing vector alone, GFD1 , gfd1 -Δ 122–143 , gfd1 -Δ 130–143 , gfd1 -Lys 135 -A-Lys 136 , or gfd1-Lys 135 -AA-Lys 136 2μ TRP1 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Trp − minimal medium plates. Plates were grown at 25, 30, and 37 °C. B , growth curve analysis of gfd1 mutants confirms that they only partially suppress rat8-2 (dbp5 ) temperature sensitivity. rat8-2 (dbp5 ) cells containing GFD1 or gfd1 mutant 2μ TRP1 test plasmids were grown to saturation and diluted, and their optical density ( OD ) was measured at A 600 for 48 h as described under “Experimental Procedures.” C , gfd1 mutants are overproduced to the same level. To examine the expression levels, rat8-2 (dbp5 ) cells containing vector alone, GFD1 , or gfd1 variant plasmids were grown at 32 °C, and whole cell lysates prepared from these cells were analyzed by immunoblotting with a polyclonal anti-Gfd1 antibody. As a loading control, Pgk1 protein levels in each lysate were detected with a monoclonal anti-Pgk1 antibody. D , gfd1 mutants with alanine insertions or deletions in the Nab2-binding α-helix do not bind to the Nab2-N. Recombinant GST-Nab2-N-wild-type was incubated with recombinant His-tagged Gfd1 wild-type (Gfd1), His-tagged gfd1 alanine insertion mutants (gfd1-Lys 135 -A-Lys 136 ; gfd1-Lys 135 -AA-Lys 136 ), or His-tagged gfd1 deletion mutant (gfd1-Δ130–143) and glutathione-Sepharose beads as described under “Experimental Procedures.” Bound fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Unbound fractions were analyzed by immunoblotting with anti-His antibody. Input recombinant His-tagged Gfd1 proteins were analyzed by SDS-PAGE and Coomassie Blue staining.
Figure Legend Snippet: gfd1 mutants that are impaired for binding to Nab2 show only partial suppression of rat8-2 (dbp5 ) temperature-sensitive growth. A , gfd1 mutants with alanine insertions ( gfd1-Lys 135 -A-Lys 136 ; gfd1-Lys 135 -AA-Lys 136 ) or deletions ( gfd1 -Δ 122–143 ; gfd -Δ 130–143 ) in the Nab2-binding α-helix only partially suppress the temperature-sensitive growth of rat8-2 (dbp5 ) cells. rat8-2 (dbp5 ) cells containing vector alone, GFD1 , gfd1 -Δ 122–143 , gfd1 -Δ 130–143 , gfd1 -Lys 135 -A-Lys 136 , or gfd1-Lys 135 -AA-Lys 136 2μ TRP1 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Trp − minimal medium plates. Plates were grown at 25, 30, and 37 °C. B , growth curve analysis of gfd1 mutants confirms that they only partially suppress rat8-2 (dbp5 ) temperature sensitivity. rat8-2 (dbp5 ) cells containing GFD1 or gfd1 mutant 2μ TRP1 test plasmids were grown to saturation and diluted, and their optical density ( OD ) was measured at A 600 for 48 h as described under “Experimental Procedures.” C , gfd1 mutants are overproduced to the same level. To examine the expression levels, rat8-2 (dbp5 ) cells containing vector alone, GFD1 , or gfd1 variant plasmids were grown at 32 °C, and whole cell lysates prepared from these cells were analyzed by immunoblotting with a polyclonal anti-Gfd1 antibody. As a loading control, Pgk1 protein levels in each lysate were detected with a monoclonal anti-Pgk1 antibody. D , gfd1 mutants with alanine insertions or deletions in the Nab2-binding α-helix do not bind to the Nab2-N. Recombinant GST-Nab2-N-wild-type was incubated with recombinant His-tagged Gfd1 wild-type (Gfd1), His-tagged gfd1 alanine insertion mutants (gfd1-Lys 135 -A-Lys 136 ; gfd1-Lys 135 -AA-Lys 136 ), or His-tagged gfd1 deletion mutant (gfd1-Δ130–143) and glutathione-Sepharose beads as described under “Experimental Procedures.” Bound fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Unbound fractions were analyzed by immunoblotting with anti-His antibody. Input recombinant His-tagged Gfd1 proteins were analyzed by SDS-PAGE and Coomassie Blue staining.

Techniques Used: Binding Assay, Plasmid Preparation, Mutagenesis, Expressing, Variant Assay, Recombinant, Incubation, SDS Page, Staining

69) Product Images from "Structural Basis for the Function of the Saccharomyces cerevisiae Gfd1 Protein in mRNA Nuclear Export *"

Article Title: Structural Basis for the Function of the Saccharomyces cerevisiae Gfd1 Protein in mRNA Nuclear Export *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.107276

nab2-Y34A shows impaired binding to Gfd1 and interacts genetically with rat8-2 (dbp5 ). A , the Nab2-N mutant nab2-Y34A does not interact with Gfd1 in vitro , but Nab2-N mutants L55W and F56D retain their ability to bind Gfd1 in vitro . Recombinant GST, GST-Nab2-N-wild-type (WT), or GST-Nab2-N mutant (Y34A, L55W, or F56D) was incubated with recombinant His-tagged Gfd1 (His-Gfd1) and glutathione-Sepharose beads as described under “Experimental Procedures.” Bound fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Unbound fractions were analyzed by immunoblotting with anti-His antibody. B , nab2-Y34A cells are viable and grow similarly to wild-type NAB2 cells. nab2 Δ cells maintained by a NAB2 URA3 plasmid and containing vector alone, NAB2 , nab2 -Δ N , nab2-Y34A , nab2-L55W , or nab2-F56D LEU2 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on control and 5-fluoroorotic acid ( 5-FOA ) plates. Cells were grown at 25, 30, and 37 °C. C , rat8-2 (dbp5 ) nab2-Y34A cells have a slow growth phenotype. nab2 Δ rat8-2 (dbp5 ) cells containing NAB2 , nab2-Y34A , nab2-L55W , or nab2-F56D LEU2 test plasmids as the sole copy of NAB2 were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Leu − minimal medium plates. Cells were grown at 25, 30, and, 32 °C. D , growth curve analysis of rat8-2 (dbp5) nab2-Y34A cells confirms that they grow more slowly than rat8-2 (dbp5) NAB2 cells. rat8-2 (dbp5 ) nab2 Δ cells carrying NAB2 , nab2-Y34A , nab2-L55W , or nab2-F56D plasmids as the sole copy of Nab2 were grown to saturation and diluted, and their optical density ( OD ) was measured at A 600 for 46 h as described under “Experimental Procedures.” E , nab2 mutants are expressed at similar levels. To examine the protein level of each nab2 variant, rat8-2 (dbp5 ) cells containing NAB2 or nab2 variant plasmids were grown at 30 °C, and whole cell lysates prepared from these cells were analyzed by immunoblotting with a polyclonal anti-Nab2 antibody. As a loading control, Pgk1 protein levels in each lysate were detected with a monoclonal anti-Pgk1 antibody. The nab2-Y34A protein level was comparable with that seen with wild-type and, although the levels of the two controls, nab2-L55W and nab2-F56D, were a little lower, this did not produce any phenotype. F , overexpression of GFD1 suppresses the slow growth phenotype of rat8-2 (dbp5) nab2-Y34A cells. nab2 Δ rat8-2 (dbp5 ) cells containing NAB2 or nab2-Y34A LEU2 plasmids as the sole copy of NAB2 and vector alone or GFD1 TRP1 plasmid were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Leu − Trp − minimal medium plates. Cells were grown at 25, 30, and, 32 °C.
Figure Legend Snippet: nab2-Y34A shows impaired binding to Gfd1 and interacts genetically with rat8-2 (dbp5 ). A , the Nab2-N mutant nab2-Y34A does not interact with Gfd1 in vitro , but Nab2-N mutants L55W and F56D retain their ability to bind Gfd1 in vitro . Recombinant GST, GST-Nab2-N-wild-type (WT), or GST-Nab2-N mutant (Y34A, L55W, or F56D) was incubated with recombinant His-tagged Gfd1 (His-Gfd1) and glutathione-Sepharose beads as described under “Experimental Procedures.” Bound fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Unbound fractions were analyzed by immunoblotting with anti-His antibody. B , nab2-Y34A cells are viable and grow similarly to wild-type NAB2 cells. nab2 Δ cells maintained by a NAB2 URA3 plasmid and containing vector alone, NAB2 , nab2 -Δ N , nab2-Y34A , nab2-L55W , or nab2-F56D LEU2 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on control and 5-fluoroorotic acid ( 5-FOA ) plates. Cells were grown at 25, 30, and 37 °C. C , rat8-2 (dbp5 ) nab2-Y34A cells have a slow growth phenotype. nab2 Δ rat8-2 (dbp5 ) cells containing NAB2 , nab2-Y34A , nab2-L55W , or nab2-F56D LEU2 test plasmids as the sole copy of NAB2 were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Leu − minimal medium plates. Cells were grown at 25, 30, and, 32 °C. D , growth curve analysis of rat8-2 (dbp5) nab2-Y34A cells confirms that they grow more slowly than rat8-2 (dbp5) NAB2 cells. rat8-2 (dbp5 ) nab2 Δ cells carrying NAB2 , nab2-Y34A , nab2-L55W , or nab2-F56D plasmids as the sole copy of Nab2 were grown to saturation and diluted, and their optical density ( OD ) was measured at A 600 for 46 h as described under “Experimental Procedures.” E , nab2 mutants are expressed at similar levels. To examine the protein level of each nab2 variant, rat8-2 (dbp5 ) cells containing NAB2 or nab2 variant plasmids were grown at 30 °C, and whole cell lysates prepared from these cells were analyzed by immunoblotting with a polyclonal anti-Nab2 antibody. As a loading control, Pgk1 protein levels in each lysate were detected with a monoclonal anti-Pgk1 antibody. The nab2-Y34A protein level was comparable with that seen with wild-type and, although the levels of the two controls, nab2-L55W and nab2-F56D, were a little lower, this did not produce any phenotype. F , overexpression of GFD1 suppresses the slow growth phenotype of rat8-2 (dbp5) nab2-Y34A cells. nab2 Δ rat8-2 (dbp5 ) cells containing NAB2 or nab2-Y34A LEU2 plasmids as the sole copy of NAB2 and vector alone or GFD1 TRP1 plasmid were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Leu − Trp − minimal medium plates. Cells were grown at 25, 30, and, 32 °C.

Techniques Used: Binding Assay, Mutagenesis, In Vitro, Recombinant, Incubation, SDS Page, Staining, Plasmid Preparation, Variant Assay, Over Expression

gfd1 mutants that are impaired for binding to Nab2 show only partial suppression of rat8-2 (dbp5 ) temperature-sensitive growth. A , gfd1 mutants with alanine insertions ( gfd1-Lys 135 -A-Lys 136 ; gfd1-Lys 135 -AA-Lys 136 ) or deletions ( gfd1 -Δ 122–143 ; gfd -Δ 130–143 ) in the Nab2-binding α-helix only partially suppress the temperature-sensitive growth of rat8-2 (dbp5 ) cells. rat8-2 (dbp5 ) cells containing vector alone, GFD1 , gfd1 -Δ 122–143 , gfd1 -Δ 130–143 , gfd1 -Lys 135 -A-Lys 136 , or gfd1-Lys 135 -AA-Lys 136 2μ TRP1 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Trp − minimal medium plates. Plates were grown at 25, 30, and 37 °C. B , growth curve analysis of gfd1 mutants confirms that they only partially suppress rat8-2 (dbp5 ) temperature sensitivity. rat8-2 (dbp5 ) cells containing GFD1 or gfd1 mutant 2μ TRP1 test plasmids were grown to saturation and diluted, and their optical density ( OD ) was measured at A 600 for 48 h as described under “Experimental Procedures.” C , gfd1 mutants are overproduced to the same level. To examine the expression levels, rat8-2 (dbp5 ) cells containing vector alone, GFD1 , or gfd1 variant plasmids were grown at 32 °C, and whole cell lysates prepared from these cells were analyzed by immunoblotting with a polyclonal anti-Gfd1 antibody. As a loading control, Pgk1 protein levels in each lysate were detected with a monoclonal anti-Pgk1 antibody. D , gfd1 mutants with alanine insertions or deletions in the Nab2-binding α-helix do not bind to the Nab2-N. Recombinant GST-Nab2-N-wild-type was incubated with recombinant His-tagged Gfd1 wild-type (Gfd1), His-tagged gfd1 alanine insertion mutants (gfd1-Lys 135 -A-Lys 136 ; gfd1-Lys 135 -AA-Lys 136 ), or His-tagged gfd1 deletion mutant (gfd1-Δ130–143) and glutathione-Sepharose beads as described under “Experimental Procedures.” Bound fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Unbound fractions were analyzed by immunoblotting with anti-His antibody. Input recombinant His-tagged Gfd1 proteins were analyzed by SDS-PAGE and Coomassie Blue staining.
Figure Legend Snippet: gfd1 mutants that are impaired for binding to Nab2 show only partial suppression of rat8-2 (dbp5 ) temperature-sensitive growth. A , gfd1 mutants with alanine insertions ( gfd1-Lys 135 -A-Lys 136 ; gfd1-Lys 135 -AA-Lys 136 ) or deletions ( gfd1 -Δ 122–143 ; gfd -Δ 130–143 ) in the Nab2-binding α-helix only partially suppress the temperature-sensitive growth of rat8-2 (dbp5 ) cells. rat8-2 (dbp5 ) cells containing vector alone, GFD1 , gfd1 -Δ 122–143 , gfd1 -Δ 130–143 , gfd1 -Lys 135 -A-Lys 136 , or gfd1-Lys 135 -AA-Lys 136 2μ TRP1 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on Trp − minimal medium plates. Plates were grown at 25, 30, and 37 °C. B , growth curve analysis of gfd1 mutants confirms that they only partially suppress rat8-2 (dbp5 ) temperature sensitivity. rat8-2 (dbp5 ) cells containing GFD1 or gfd1 mutant 2μ TRP1 test plasmids were grown to saturation and diluted, and their optical density ( OD ) was measured at A 600 for 48 h as described under “Experimental Procedures.” C , gfd1 mutants are overproduced to the same level. To examine the expression levels, rat8-2 (dbp5 ) cells containing vector alone, GFD1 , or gfd1 variant plasmids were grown at 32 °C, and whole cell lysates prepared from these cells were analyzed by immunoblotting with a polyclonal anti-Gfd1 antibody. As a loading control, Pgk1 protein levels in each lysate were detected with a monoclonal anti-Pgk1 antibody. D , gfd1 mutants with alanine insertions or deletions in the Nab2-binding α-helix do not bind to the Nab2-N. Recombinant GST-Nab2-N-wild-type was incubated with recombinant His-tagged Gfd1 wild-type (Gfd1), His-tagged gfd1 alanine insertion mutants (gfd1-Lys 135 -A-Lys 136 ; gfd1-Lys 135 -AA-Lys 136 ), or His-tagged gfd1 deletion mutant (gfd1-Δ130–143) and glutathione-Sepharose beads as described under “Experimental Procedures.” Bound fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Unbound fractions were analyzed by immunoblotting with anti-His antibody. Input recombinant His-tagged Gfd1 proteins were analyzed by SDS-PAGE and Coomassie Blue staining.

Techniques Used: Binding Assay, Plasmid Preparation, Mutagenesis, Expressing, Variant Assay, Recombinant, Incubation, SDS Page, Staining

70) Product Images from "Importin 13 Mediates Nuclear Import of Histone Fold-containing Chromatin Accessibility Complex Heterodimers"

Article Title: Importin 13 Mediates Nuclear Import of Histone Fold-containing Chromatin Accessibility Complex Heterodimers

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M806820200

Monomeric CHRAC-15 and CHRAC-17 are not imported by importin 13. The HeLa P4 cells were transiently transfected with plasmid DNA encoding CHRAC-15 ( A ) and CHRAC-17 ( B ) N-terminally fused either to EGFP or RFP. FLAG-tagged importin 13 was additionally coexpressed as indicated. The subcellular distribution of the transfected fusion proteins was determined by direct fluorescence 24 h post-transfection. The DNA was counterstained with Hoechst. Although EGFP-CHRAC-15 shows a homogenous distribution in transfected cells at steady state, RFP-CHRAC-15 and fluorescently labeled CHRAC-17 are localized in the cytoplasm. The coexpression of FLAG-importin 13 did not change the subcellular distribution of the fluorescently labeled CHRAC subunits. C , GST-tagged CHRAC-15 and CHRAC-17 were expressed in E. coli , immobilized on glutathione-Sepharose, and incubated with recombinant purified FLAG-tagged importin 13 in the absence (-) or presence (+) of RanGTP (Q69L mutant). The input of importin 13 represents 20% of the amount used in the assay. Bound fractions were analyzed by SDS-PAGE followed by Coomassie staining. Importin 13 does not bind efficiently to the monomeric CHRAC subunits. Mw , molecular mass; imp13 , importin 13.
Figure Legend Snippet: Monomeric CHRAC-15 and CHRAC-17 are not imported by importin 13. The HeLa P4 cells were transiently transfected with plasmid DNA encoding CHRAC-15 ( A ) and CHRAC-17 ( B ) N-terminally fused either to EGFP or RFP. FLAG-tagged importin 13 was additionally coexpressed as indicated. The subcellular distribution of the transfected fusion proteins was determined by direct fluorescence 24 h post-transfection. The DNA was counterstained with Hoechst. Although EGFP-CHRAC-15 shows a homogenous distribution in transfected cells at steady state, RFP-CHRAC-15 and fluorescently labeled CHRAC-17 are localized in the cytoplasm. The coexpression of FLAG-importin 13 did not change the subcellular distribution of the fluorescently labeled CHRAC subunits. C , GST-tagged CHRAC-15 and CHRAC-17 were expressed in E. coli , immobilized on glutathione-Sepharose, and incubated with recombinant purified FLAG-tagged importin 13 in the absence (-) or presence (+) of RanGTP (Q69L mutant). The input of importin 13 represents 20% of the amount used in the assay. Bound fractions were analyzed by SDS-PAGE followed by Coomassie staining. Importin 13 does not bind efficiently to the monomeric CHRAC subunits. Mw , molecular mass; imp13 , importin 13.

Techniques Used: Transfection, Plasmid Preparation, Fluorescence, Labeling, Incubation, Recombinant, Purification, Mutagenesis, SDS Page, Staining

Basic amino acids in the CHRAC-15/17 subunits are necessary for importin 13 binding. A , HeLa P4 cells were transiently cotransfected with plasmid DNA coding for wt EGFP-CHRAC-15 and RFP-CHRAC-17 ( top row ) and fusion proteins carrying an increasing number of mutated basic amino acid residues as indicated (see also supplemental Fig. S5). FLAG-importin 13 was coexpressed in each approach. The subcellular localization of wild type and mutated CHRAC-15/17 complexes was determined by direct fluorescence 24 h post-transfection. The colocalization of RFP and EGFP fusion proteins is shown in yellow (merge). DNA was counterstained with Hoechst. The stepwise substitution of basic amino acids in CHRAC-15 and CHRAC-17 interferes with the importin 13-mediated nuclear transport and results in an increased cytoplasmic accumulation of the CHRAC-15/17 complex in transfected cells. B , for quantitative analysis, the fluorescence intensity of colocalized wild type and mutated CHRAC-15/17 complexes was measured in 20 cells using the ImageJ software (National Institutes of Health). The percentage of nuclear ( nucl. ) and cytoplasmic ( cyto. ) localization was calculated. Sites of mutations are indicated with Mut1, Mut2, Mut3, and Mut4 as in A. C , GST-CHRAC-15/His-CHRAC-17 with alanine substitutions of conserved basic amino acids were coexpressed in E. coli and immobilized on glutathione-Sepharose. Binding studies with the wt and the mutated heterodimers (described above) were performed using recombinant purified importin 13. 20% of importin 13 used in this assay are displayed as input. Each indicated amino acid substitution is additional compared with the preceding column. To imitate the RanGTP gradient between the cytoplasmic and the nuclear compartment, the binding was also performed in the presence (+) of RanGTP (Q69L mutant). After incubation, the bound fractions were displayed by SDS-PAGE and stained by Coomassie. The loss of positively charged amino acids in the CHRAC-15/17 complex results in a reduced binding of importin 13. Mw , molecular mass; imp13 , importin 13.
Figure Legend Snippet: Basic amino acids in the CHRAC-15/17 subunits are necessary for importin 13 binding. A , HeLa P4 cells were transiently cotransfected with plasmid DNA coding for wt EGFP-CHRAC-15 and RFP-CHRAC-17 ( top row ) and fusion proteins carrying an increasing number of mutated basic amino acid residues as indicated (see also supplemental Fig. S5). FLAG-importin 13 was coexpressed in each approach. The subcellular localization of wild type and mutated CHRAC-15/17 complexes was determined by direct fluorescence 24 h post-transfection. The colocalization of RFP and EGFP fusion proteins is shown in yellow (merge). DNA was counterstained with Hoechst. The stepwise substitution of basic amino acids in CHRAC-15 and CHRAC-17 interferes with the importin 13-mediated nuclear transport and results in an increased cytoplasmic accumulation of the CHRAC-15/17 complex in transfected cells. B , for quantitative analysis, the fluorescence intensity of colocalized wild type and mutated CHRAC-15/17 complexes was measured in 20 cells using the ImageJ software (National Institutes of Health). The percentage of nuclear ( nucl. ) and cytoplasmic ( cyto. ) localization was calculated. Sites of mutations are indicated with Mut1, Mut2, Mut3, and Mut4 as in A. C , GST-CHRAC-15/His-CHRAC-17 with alanine substitutions of conserved basic amino acids were coexpressed in E. coli and immobilized on glutathione-Sepharose. Binding studies with the wt and the mutated heterodimers (described above) were performed using recombinant purified importin 13. 20% of importin 13 used in this assay are displayed as input. Each indicated amino acid substitution is additional compared with the preceding column. To imitate the RanGTP gradient between the cytoplasmic and the nuclear compartment, the binding was also performed in the presence (+) of RanGTP (Q69L mutant). After incubation, the bound fractions were displayed by SDS-PAGE and stained by Coomassie. The loss of positively charged amino acids in the CHRAC-15/17 complex results in a reduced binding of importin 13. Mw , molecular mass; imp13 , importin 13.

Techniques Used: Binding Assay, Plasmid Preparation, Fluorescence, Transfection, Software, Recombinant, Purification, Mutagenesis, Incubation, SDS Page, Staining

71) Product Images from "Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope"

Article Title: Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200805049

ERK1/2 interacts with lamin A in vitro and in vivo. (A, B) Lysates from HEK293 cells (stimulated 5 min with EGF after serum deprivation) were immunoprecipitated with either anti-lamin A/C (sc-20680), anti-ERK2 or anti-Sp1 antibodies. Preimmune serum was used as control. Both the input and the whole immunoprecipitated material were subjected to Western blot analysis using anti-ERK2, anti-pERK1/2, anti-Sp1 and anti-lamin A/C (sc-20680) antibodies as indicated. (C) GST-lamin A fusion proteins containing amino acid residues 37–244, 243–388 or 453–571 of rat lamin A were tested for their interaction with endogenous ERK1/2 from whole HEK293 cell extracts. Approximately 90% of the reaction mixture was precipitated with gluthatione-Sepharose 4B, washed and analyzed by Western blot. (D) Lysates from HEK293 cells overexpressing FLAG-tagged ERK2 (wild-type and several mutants) were immunoprecipitated with an anti-FLAG antibody. Both the input and the whole immunoprecipitated material were subjected to Western blot analysis using anti-FLAG and anti-lamin A/C (sc-20680) antibodies. In the scheme in C and D, (+) and (−) indicate positive and negative interactions, respectively. NLS: Nuclear localization signal. Numbers on the side of the blots indicate molecular weights (in kD).
Figure Legend Snippet: ERK1/2 interacts with lamin A in vitro and in vivo. (A, B) Lysates from HEK293 cells (stimulated 5 min with EGF after serum deprivation) were immunoprecipitated with either anti-lamin A/C (sc-20680), anti-ERK2 or anti-Sp1 antibodies. Preimmune serum was used as control. Both the input and the whole immunoprecipitated material were subjected to Western blot analysis using anti-ERK2, anti-pERK1/2, anti-Sp1 and anti-lamin A/C (sc-20680) antibodies as indicated. (C) GST-lamin A fusion proteins containing amino acid residues 37–244, 243–388 or 453–571 of rat lamin A were tested for their interaction with endogenous ERK1/2 from whole HEK293 cell extracts. Approximately 90% of the reaction mixture was precipitated with gluthatione-Sepharose 4B, washed and analyzed by Western blot. (D) Lysates from HEK293 cells overexpressing FLAG-tagged ERK2 (wild-type and several mutants) were immunoprecipitated with an anti-FLAG antibody. Both the input and the whole immunoprecipitated material were subjected to Western blot analysis using anti-FLAG and anti-lamin A/C (sc-20680) antibodies. In the scheme in C and D, (+) and (−) indicate positive and negative interactions, respectively. NLS: Nuclear localization signal. Numbers on the side of the blots indicate molecular weights (in kD).

Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Western Blot

ERK1/2-dependent phosphorylation of c-Fos affects its interaction with lamin A. GST-lamin A fusion protein containing amino acid residues 37–244 of rat lamin A was tested for its interaction with either endogenous c-Fos present in the SNF of NIH-3T3 cells (A and B) or with c-Fos-wt or c-Fos-m ectopically expressed in U2OS cells (C). Nuclear extracts were incubated with GST proteins and 1/20 of the reaction mixture was examined by Western blot (INPUT). The rest was precipitated with gluthatione-Sepharose 4B, washed and analyzed by Western blot. The used antibodies and the detected proteins are indicated. Numbers on the side of the blots indicate molecular weight (in kD). In B, cells were subjected to the protocol schematized in Fig. 2 A . In C, cells were transfected with c-Fos-wt or c-Fos-m. 24 h after transfection, cells were starved for 24 h and stimulated with serum for 30 min.
Figure Legend Snippet: ERK1/2-dependent phosphorylation of c-Fos affects its interaction with lamin A. GST-lamin A fusion protein containing amino acid residues 37–244 of rat lamin A was tested for its interaction with either endogenous c-Fos present in the SNF of NIH-3T3 cells (A and B) or with c-Fos-wt or c-Fos-m ectopically expressed in U2OS cells (C). Nuclear extracts were incubated with GST proteins and 1/20 of the reaction mixture was examined by Western blot (INPUT). The rest was precipitated with gluthatione-Sepharose 4B, washed and analyzed by Western blot. The used antibodies and the detected proteins are indicated. Numbers on the side of the blots indicate molecular weight (in kD). In B, cells were subjected to the protocol schematized in Fig. 2 A . In C, cells were transfected with c-Fos-wt or c-Fos-m. 24 h after transfection, cells were starved for 24 h and stimulated with serum for 30 min.

Techniques Used: Incubation, Western Blot, Molecular Weight, Transfection

72) Product Images from "Starch Biosynthetic Enzymes from Developing Maize Endosperm Associate in Multisubunit Complexes 1Starch Biosynthetic Enzymes from Developing Maize Endosperm Associate in Multisubunit Complexes 1 [OA]"

Article Title: Starch Biosynthetic Enzymes from Developing Maize Endosperm Associate in Multisubunit Complexes 1Starch Biosynthetic Enzymes from Developing Maize Endosperm Associate in Multisubunit Complexes 1 [OA]

Journal: Plant Physiology

doi: 10.1104/pp.108.116285

Mass spectrometry identification of affinity purified proteins. Amyloplast proteins were fractionated by affinity chromatography using S-protein agarose coupled to either SSI or BEIIa as the matrix. A, Sypro Ruby stain. M lanes contain molecular mass
Figure Legend Snippet: Mass spectrometry identification of affinity purified proteins. Amyloplast proteins were fractionated by affinity chromatography using S-protein agarose coupled to either SSI or BEIIa as the matrix. A, Sypro Ruby stain. M lanes contain molecular mass

Techniques Used: Mass Spectrometry, Affinity Purification, Affinity Chromatography, Staining

Affinity chromatography using amyloplast extracts. Amyloplast extracts in low salt buffer were incubated with S-protein agarose attached to the indicated recombinant maize proteins or S-protein agarose without any recombinant protein attached (indicated
Figure Legend Snippet: Affinity chromatography using amyloplast extracts. Amyloplast extracts in low salt buffer were incubated with S-protein agarose attached to the indicated recombinant maize proteins or S-protein agarose without any recombinant protein attached (indicated

Techniques Used: Affinity Chromatography, Incubation, Recombinant

73) Product Images from "Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export *Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export * S⃞Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export * S⃞ "

Article Title: Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export *Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export * S⃞Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M803649200

The Nab2-binding domain of Mlp1 directly interacts with Nab2 and causes nuclear accumulation of poly(A) RNA. A , the Nab2-binding domain of Mlp1 (Mlp1-NBD; residues 1586–1768) binds to the N-terminal domain of Nab2 (Nab2-N; residues 1–97) in vitro . His-Nab2-N (Nab2-N) or, as a control, ovalbumin ( Oval .) coupled to Sepharose beads was incubated with His-Mlp1-NBD and bound fractions were analyzed by SDS-PAGE and Coomassie staining. B , the Nab2-binding domain of Mlp1 (Mlp1-NBD) co-purifies with the N-terminal domain of Nab2 (Nab2-N). Cell lysates from E. coli expressing His-Mlp1-NBD and E. coli expressing GST-Nab2-N were combined, incubated with glutathione beads, and beads were washed three times. Input combined lysate ( I ), unbound ( U ) fraction, three wash fractions ( W1, W2, W3 ), and bound ( B ) fraction were analyzed by SDS-PAGE and Coomassie staining. C , expression of the Nab2-binding domain of Mlp1 ( Mlp1-NBD ) relocalizes ΔRGG-Nab2-GFP to the nucleus. CT-Mlp1 and Mlp1-NBD were expressed in yeast cells expressing ΔRGG-Nab2-GFP, which displays localization throughout the cell ( 36 ), and ΔRGG-Nab2-GFP was visualized by direct fluorescence microscopy. Empty vector ( Vec ) was used as control. Corresponding differential interference contrast ( DIC ) images are shown. D , expression of the Nab2-binding domain of Mlp1 (Mlp1-NBD) causes nuclear accumulation of poly(A) RNA. CT-Mlp1 and Mlp1-NBD were expressed in yeast cells and poly(A) RNA ( Poly ( A )) was visualized by fluorescence in situ hybridization with an oligo(dT) probe as described under “Experimental Procedures.” Cells were stained with DAPI to visualize the position of the nucleus. Corresponding differential interference contrast images are shown.
Figure Legend Snippet: The Nab2-binding domain of Mlp1 directly interacts with Nab2 and causes nuclear accumulation of poly(A) RNA. A , the Nab2-binding domain of Mlp1 (Mlp1-NBD; residues 1586–1768) binds to the N-terminal domain of Nab2 (Nab2-N; residues 1–97) in vitro . His-Nab2-N (Nab2-N) or, as a control, ovalbumin ( Oval .) coupled to Sepharose beads was incubated with His-Mlp1-NBD and bound fractions were analyzed by SDS-PAGE and Coomassie staining. B , the Nab2-binding domain of Mlp1 (Mlp1-NBD) co-purifies with the N-terminal domain of Nab2 (Nab2-N). Cell lysates from E. coli expressing His-Mlp1-NBD and E. coli expressing GST-Nab2-N were combined, incubated with glutathione beads, and beads were washed three times. Input combined lysate ( I ), unbound ( U ) fraction, three wash fractions ( W1, W2, W3 ), and bound ( B ) fraction were analyzed by SDS-PAGE and Coomassie staining. C , expression of the Nab2-binding domain of Mlp1 ( Mlp1-NBD ) relocalizes ΔRGG-Nab2-GFP to the nucleus. CT-Mlp1 and Mlp1-NBD were expressed in yeast cells expressing ΔRGG-Nab2-GFP, which displays localization throughout the cell ( 36 ), and ΔRGG-Nab2-GFP was visualized by direct fluorescence microscopy. Empty vector ( Vec ) was used as control. Corresponding differential interference contrast ( DIC ) images are shown. D , expression of the Nab2-binding domain of Mlp1 (Mlp1-NBD) causes nuclear accumulation of poly(A) RNA. CT-Mlp1 and Mlp1-NBD were expressed in yeast cells and poly(A) RNA ( Poly ( A )) was visualized by fluorescence in situ hybridization with an oligo(dT) probe as described under “Experimental Procedures.” Cells were stained with DAPI to visualize the position of the nucleus. Corresponding differential interference contrast images are shown.

Techniques Used: Binding Assay, In Vitro, Incubation, SDS Page, Staining, Expressing, Fluorescence, Microscopy, Plasmid Preparation, In Situ Hybridization

Mapping of CT-Mlp1 reveals a Nab2-binding region between residues 1586 and 1779. A , schematic depicting sizes of CT-Mlp1 truncation mutants ( CT1–7 ) and the Nab2-binding domain of Mlp1 ( Mlp1-NBD ) constructed. B , CT-Mlp1 truncation mutant CT2 (residues 1490–1779) binds to Nab2 in vitro , but mutant CT5 (residues 1490–1585) does not bind to Nab2. Recombinant GST, GST-CT-Mlp1 ( CT-Mlp1 ), or the GST-CT-Mlp1 truncation mutant ( CT2, CT4, CT5 ) bound to glutathione-Sepharose beads were incubated with recombinant His-tagged Nab2 ( Nab2 ) and unbound ( U ) and bound ( B ) fractions were analyzed by immunoblotting with anti-His antibody. The percentage of His-Nab2 band intensity in each bound fraction relative to the His-Nab2 band intensity in the bound fraction of GST-CT-Mlp1 is indicated below the immunoblot. C , CT-Mlp1 truncation mutant CT6 (residues 1586–1779) binds to Nab2 in vitro . Recombinant GST, GST-CT-Mlp1 ( CT-Mlp1 ), or the GST-CT-Mlp1 truncation mutant ( CT6, CT7 ) bound to glutathione-Sepharose beads was incubated with recombinant His-tagged Nab2 ( Nab2 ) and unbound ( U ) and bound ( B ) fractions were analyzed by immunoblotting with anti-His antibody. The percentage of His-Nab2 band intensity in each bound fraction relative to the His-Nab2 band intensity in the bound fraction of GST-CT-Mlp1 is indicated below the immunoblot. D , expression of CT-Mlp1 truncation mutant CT2 (residues 1490–1779) causes relocalization of ΔRGG-Nab2-GFP to the nucleus. CT-Mlp1 and CT-Mlp1 truncation mutants ( CT1–5 ) were expressed in yeast cells expressing a Nab2 mutant fused to GFP (ΔRGG-Nab2-GFP), which displays localization throughout the cell ( 36 ), and ΔRGG-Nab2-GFP was visualized by direct fluorescence microscopy. Empty vector ( Vec ) was used as a control. Corresponding differential interference contrast ( DIC ) images are shown.
Figure Legend Snippet: Mapping of CT-Mlp1 reveals a Nab2-binding region between residues 1586 and 1779. A , schematic depicting sizes of CT-Mlp1 truncation mutants ( CT1–7 ) and the Nab2-binding domain of Mlp1 ( Mlp1-NBD ) constructed. B , CT-Mlp1 truncation mutant CT2 (residues 1490–1779) binds to Nab2 in vitro , but mutant CT5 (residues 1490–1585) does not bind to Nab2. Recombinant GST, GST-CT-Mlp1 ( CT-Mlp1 ), or the GST-CT-Mlp1 truncation mutant ( CT2, CT4, CT5 ) bound to glutathione-Sepharose beads were incubated with recombinant His-tagged Nab2 ( Nab2 ) and unbound ( U ) and bound ( B ) fractions were analyzed by immunoblotting with anti-His antibody. The percentage of His-Nab2 band intensity in each bound fraction relative to the His-Nab2 band intensity in the bound fraction of GST-CT-Mlp1 is indicated below the immunoblot. C , CT-Mlp1 truncation mutant CT6 (residues 1586–1779) binds to Nab2 in vitro . Recombinant GST, GST-CT-Mlp1 ( CT-Mlp1 ), or the GST-CT-Mlp1 truncation mutant ( CT6, CT7 ) bound to glutathione-Sepharose beads was incubated with recombinant His-tagged Nab2 ( Nab2 ) and unbound ( U ) and bound ( B ) fractions were analyzed by immunoblotting with anti-His antibody. The percentage of His-Nab2 band intensity in each bound fraction relative to the His-Nab2 band intensity in the bound fraction of GST-CT-Mlp1 is indicated below the immunoblot. D , expression of CT-Mlp1 truncation mutant CT2 (residues 1490–1779) causes relocalization of ΔRGG-Nab2-GFP to the nucleus. CT-Mlp1 and CT-Mlp1 truncation mutants ( CT1–5 ) were expressed in yeast cells expressing a Nab2 mutant fused to GFP (ΔRGG-Nab2-GFP), which displays localization throughout the cell ( 36 ), and ΔRGG-Nab2-GFP was visualized by direct fluorescence microscopy. Empty vector ( Vec ) was used as a control. Corresponding differential interference contrast ( DIC ) images are shown.

Techniques Used: Binding Assay, Construct, Mutagenesis, In Vitro, Recombinant, Incubation, Expressing, Fluorescence, Microscopy, Plasmid Preparation

74) Product Images from "Dematin, a Component of the Erythrocyte Membrane Skeleton, Is Internalized by the Malaria Parasite and Associates with Plasmodium 14-3-3 *"

Article Title: Dematin, a Component of the Erythrocyte Membrane Skeleton, Is Internalized by the Malaria Parasite and Associates with Plasmodium 14-3-3 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.194613

Erythroid dematin directly interacts with Pb14-3-3. A , glutathione-Sepharose-immobilized GST-Pb14-3-3 or GST as a control was incubated with a P. berghei extract solubilized by sonication. The eluted proteins were subjected to Western blot analysis using an α-dematin mAb. B , dematin was immunopurified from P. berghei parasites using the specific α-dematin mAb. Parasite extract (input, 1/100), pretreated with protein-G beads (preclearing, 1/20), was incubated with α-dematin-conjugated protein-G beads. After removal of the unbound material (flow-through, 1/100), immunoprecipitated proteins were eluted ( IP , 1/20). Blotted samples were probed with α-dematin mAb and α-Pb14-3-3. The overlay assay was performed using GST-Pb14-3-3 as a probe. The interactions were inhibited through the addition of the A8Ap phosphopeptide. Asterisks in both panels indicate the bands corresponding to the 48- and 52-kDa dematin.
Figure Legend Snippet: Erythroid dematin directly interacts with Pb14-3-3. A , glutathione-Sepharose-immobilized GST-Pb14-3-3 or GST as a control was incubated with a P. berghei extract solubilized by sonication. The eluted proteins were subjected to Western blot analysis using an α-dematin mAb. B , dematin was immunopurified from P. berghei parasites using the specific α-dematin mAb. Parasite extract (input, 1/100), pretreated with protein-G beads (preclearing, 1/20), was incubated with α-dematin-conjugated protein-G beads. After removal of the unbound material (flow-through, 1/100), immunoprecipitated proteins were eluted ( IP , 1/20). Blotted samples were probed with α-dematin mAb and α-Pb14-3-3. The overlay assay was performed using GST-Pb14-3-3 as a probe. The interactions were inhibited through the addition of the A8Ap phosphopeptide. Asterisks in both panels indicate the bands corresponding to the 48- and 52-kDa dematin.

Techniques Used: Incubation, Sonication, Western Blot, Flow Cytometry, Immunoprecipitation, Overlay Assay

Dematin-Pb14-3-3 interaction is promoted by PKA phosphorylation. A , a GST-fused 52-kDa isoform of dematin was phosphorylated in vitro using the PKA catalytic subunit or casein kinase II ( CKII ), digested with the PreScission enzyme, and run in SDS-PAGE. A control sample without enzymes was also included. Autoradiography ( Autorad. ) of the stained gel showed that the recombinant dematin was efficiently labeled only by PKA. B , glutathione-Sepharose-immobilized GST or GST-dematin, phosphorylated (+) or not (−) with PKA, was incubated with a bacterial lysate containing His-Pb14-3-3. Proteins eluted with A8Ap synthetic phosphopeptide or glutathione were blotted and probed with α-His 6 , to detect the associated 14-3-3, or α-GST, to detect the eluted recombinant GST and GST-dematin. C , putative 14-3-3-binding sites on the 52-kDa dematin isoform are underlined . Putative PKA phosphorylation sites coinciding with the 14-3-3-binding sites are in bold . The position of mutated serines is specified. The ATP-binding site (P-loop) is boxed in gray. D , recombinant GST, GST-dematin, or GST-dematin mutants, bound to glutathione-Sepharose beads, were phosphorylated by the PKA catalytic subunit. Phosphorylated recombinant proteins, digested with the PreScission enzyme, were run in SDS-PAGE, stained with Coomassie Blue, and subjected to autoradiography. E , GST, GST-dematin, and GST-dematin mutants were treated as in panel B . Proteins eluted from the glutathione-Sepharose beads with the A8Ap synthetic phosphopeptide were subjected to immunoblotting using the α-His 6 to detect bound 14-3-3.
Figure Legend Snippet: Dematin-Pb14-3-3 interaction is promoted by PKA phosphorylation. A , a GST-fused 52-kDa isoform of dematin was phosphorylated in vitro using the PKA catalytic subunit or casein kinase II ( CKII ), digested with the PreScission enzyme, and run in SDS-PAGE. A control sample without enzymes was also included. Autoradiography ( Autorad. ) of the stained gel showed that the recombinant dematin was efficiently labeled only by PKA. B , glutathione-Sepharose-immobilized GST or GST-dematin, phosphorylated (+) or not (−) with PKA, was incubated with a bacterial lysate containing His-Pb14-3-3. Proteins eluted with A8Ap synthetic phosphopeptide or glutathione were blotted and probed with α-His 6 , to detect the associated 14-3-3, or α-GST, to detect the eluted recombinant GST and GST-dematin. C , putative 14-3-3-binding sites on the 52-kDa dematin isoform are underlined . Putative PKA phosphorylation sites coinciding with the 14-3-3-binding sites are in bold . The position of mutated serines is specified. The ATP-binding site (P-loop) is boxed in gray. D , recombinant GST, GST-dematin, or GST-dematin mutants, bound to glutathione-Sepharose beads, were phosphorylated by the PKA catalytic subunit. Phosphorylated recombinant proteins, digested with the PreScission enzyme, were run in SDS-PAGE, stained with Coomassie Blue, and subjected to autoradiography. E , GST, GST-dematin, and GST-dematin mutants were treated as in panel B . Proteins eluted from the glutathione-Sepharose beads with the A8Ap synthetic phosphopeptide were subjected to immunoblotting using the α-His 6 to detect bound 14-3-3.

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

75) Product Images from "Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp"

Article Title: Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp

Journal: PLoS ONE

doi: 10.1371/journal.pone.0126987

Agarose gel of PCR amplicons from VP AHPND using the AP3 method. Lane M; DNA marker, Lane N: negative control; Lanes 1–3: Positive amplicons (333 bp) from 3 isolates of VP AHPND bacteria; Lanes 4–10: No amplicons from 8 non-AHPND bacteria; Lane P: positive control (333 bp).
Figure Legend Snippet: Agarose gel of PCR amplicons from VP AHPND using the AP3 method. Lane M; DNA marker, Lane N: negative control; Lanes 1–3: Positive amplicons (333 bp) from 3 isolates of VP AHPND bacteria; Lanes 4–10: No amplicons from 8 non-AHPND bacteria; Lane P: positive control (333 bp).

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker, Negative Control, Positive Control

Bacterial expression of ToxA and ToxB. (A) ToxA expressed with a 6-His tag and purified by Ni-NTA affinity chromatography. Lane 1: Bacterial cell lysate from a non-induced bacterial culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted protein from the Ni-NTA column. The deduced molecular weight for ToxA-His was 12.7 kDa. (B) ToxB was expressed as a GST-fusion protein. Lane 1: Bacterial cell lysate from a non-induced culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted fraction from Sepharose 4B beads; Lanes 4 5: Fraction eluted from Sepahrose 4B after thrombin-cut. The estimated molecular weights for GST-ToxB and ToxB were approximately 76 and 50 kDa, respectively.
Figure Legend Snippet: Bacterial expression of ToxA and ToxB. (A) ToxA expressed with a 6-His tag and purified by Ni-NTA affinity chromatography. Lane 1: Bacterial cell lysate from a non-induced bacterial culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted protein from the Ni-NTA column. The deduced molecular weight for ToxA-His was 12.7 kDa. (B) ToxB was expressed as a GST-fusion protein. Lane 1: Bacterial cell lysate from a non-induced culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted fraction from Sepharose 4B beads; Lanes 4 5: Fraction eluted from Sepahrose 4B after thrombin-cut. The estimated molecular weights for GST-ToxB and ToxB were approximately 76 and 50 kDa, respectively.

Techniques Used: Expressing, Purification, Affinity Chromatography, Molecular Weight

Related Articles

Centrifugation:

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

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

Filtration:

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

Expressing:

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

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

Article Title: Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13
Article Snippet: Paragraph title: Protein expression and purification. ... Recombinant proteins were purified on glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, N.J.) or Ni-nitrilotriacetic acid agarose (Qiagen).

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

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

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

Construct:

Article Title: Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13
Article Snippet: Recombinant proteins were purified on glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, N.J.) or Ni-nitrilotriacetic acid agarose (Qiagen). .. The pET41a constructs were expressed in strain BL21(DE3) Codon Plus RIL (Stratagene, La Jolla, Calif.).

Electrophoresis:

Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
Article Snippet: Before electrophoresis, output samples were centrifuged (16,000 × g , 5 min), and only the supernatant was used for immunoblotting. .. For each reaction, 20 μl (bed volume) glutathione-Sepharose 4B (GE Healthcare) loaded with GST or GST fusion proteins was washed twice in 700 μl binding buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM DTT, 10% glycerol, 0.5% Triton X-100, EDTA-free protease inhibitor cocktail set III).

Incubation:

Article Title: The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein
Article Snippet: Expression was induced by adding IPTG to 0.5 mM and the cultures were incubated for a further 4 h before harvesting the cells. .. Clarified lysates were mixed with pre-washed Ni-NTA superflow (Qiagen) or glutathione-sepharose (GE Healthcare) resin and after extensive washing with lysis buffer, the bound proteins recovered by elution in lysis buffer containing 250 mM imidazole or 20 mM reduced glutathione.

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

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

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

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿
Article Snippet: .. The GST and GST-Xic1 proteins (5 μg) were coupled to glutathione-Sepharose 4B (GE Healthcare) and incubated in 250 μl of LSS for 1 h at 4°C. .. The beads were washed extensively with NETN buffer (50 mM Tris at pH 8, 250 mM NaCl, 5 mM EDTA, and 0.5% Nonidet P-40).

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

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

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

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

Mass Spectrometry:

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

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

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

Acrylamide Gel Assay:

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

Western Blot:

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

Transformation Assay:

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

High Performance Liquid Chromatography:

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

Protease Inhibitor:

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

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

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

SDS Page:

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

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

Sonication:

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

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

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

Affinity Purification:

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

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

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

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

Recombinant:

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

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

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

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

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

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

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

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

GST Pulldown Assay:

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

Nucleic Acid Electrophoresis:

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

Ion Exchange Chromatography:

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

Purification:

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

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

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

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

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

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

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

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

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

Affinity Chromatography:

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

Blocking Assay:

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

Lysis:

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

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

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

Liquid Chromatography:

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

Binding Assay:

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

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

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

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

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

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

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

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

In Vitro:

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

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

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

Protein Binding:

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

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

Concentration Assay:

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

Staining:

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

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

    Journal: eLife

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

    doi: 10.7554/eLife.03473

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

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

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

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

    Journal: eLife

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

    doi: 10.7554/eLife.03473

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

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

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

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

    Journal: eLife

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

    doi: 10.7554/eLife.03473

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

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

    Techniques: Incubation, SDS Page, Staining, Western Blot

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

    Journal: eLife

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

    doi: 10.7554/eLife.03473

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

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

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

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

    Journal: eLife

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

    doi: 10.7554/eLife.03473

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

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

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

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

    Journal: eLife

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

    doi: 10.7554/eLife.03473

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

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

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

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

    Journal: eLife

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

    doi: 10.7554/eLife.03473

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1007117

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1007117

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1007117

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1007117

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

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

    Techniques: Mass Spectrometry, Cell Culture

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1007117

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1007117

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

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

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