anti ciap1 goat polyclonal antibody pab  (R&D Systems)

Journal: PLoS ONE

Article Title: E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

doi: 10.1371/journal.pone.0206253

Figure Lengend Snippet: GST-pull-down analysis of the interaction of E2F1 with cIAP1. GST-fusion proteins were produced in bacteria, immobilized on glutathione-sepharose and incubated in the presence of E2F1 recombinant protein (A) or HeLa cell lysate (B). The interaction was revealed by Western-blot analysis. Increasing concentrations of the Smac-N7 peptide (μg/mL) or the Smac mimetic BV6 (nM) or DMSO vehicle buffer corresponding to the higher concentration (0) were added to immobilized GST-fusion proteins 30 min before the interaction step (B). E2F1 and TRAF2 binding were quantified using Image J software and related to binding without IAC (lower panel). One representative experiment is shown.

Article Snippet: Lysates were pre-cleared using 20μl of Protein A/G+ Agarose beads (Sigma-Aldrich), and incubated overnight at 4°C with mouse IgG1 purified anti-HA.11 (Biolegend, Ozyme, Montigny-le-Bretonneux, France), goat polyclonal anti-cIAP1 antibody (R&D systems Europe, Lille, France) or mouse or goat IgG.

Techniques: Produced, Bacteria, Incubation, Recombinant, Western Blot, Concentration Assay, Binding Assay, Software

Journal: PLoS ONE

Article Title: E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

doi: 10.1371/journal.pone.0206253

Figure Lengend Snippet: (A) Primary structure of the cIAP1-BIR3 and BIR3 A-H deletion mutants. (B & C) GST-pull-down analysis of the interaction of E2F1 with the BIR3 deletion mutants described in A (B) or with wt cIAP1, isolated BIR3 or cIAP1 devoid of the BIR3 α1-helix (cIAP1Δα1) or the whole BIR3 (cIAP1ΔBIR3)(C). GST-fusion proteins were produced in bacteria, immobilized on glutathione-sepharose and incubated in the presence of E2F1 recombinant protein (B) or HeLa cell lysate (C). The interaction was revealed by Western-blot analysis. Mw: molecular weights. (D) Analysis of ubiquitination of E2F1 in HeLa cells transfected with wt 3HA-E2F1, His-Ubiquitin, an empty vector or a vector encoding wt cIAP1 or the BIR3-α1-helix deleted mutant. Ubiquitinated proteins were pulled-down using cobalt beads, and ubiquitinated E2F1 was revealed using an anti-E2F1 antibody. (E) E2F1 transcriptional activity was assessed in luciferase gene experiments using constructs in which luciferase expression was controlled by the CCNE or CAS7 promoter. Luciferase activity was normalized to β-galactosidase activity and expressed as fold-induction of promoter activity measured in cells transfected with empty vectors. The mean ± S.D. of at least 3 independent experiments is shown. Statistical analysis was performed using Student’s t test. (F) GST-pull-down analysis of the interaction of E2F1, Smac or TRAF2 with wt cIAP1, cIAP1 devoid of the BIR3 α1-helix (cIAP1Δα1) or cIAP1 altered in the TRAF2 interacting site (cIAP1-L47A). GST-fusion proteins were produced in bacteria, immobilized on glutathione-sepharose and incubated in the presence of HeLa cell lysate. The interaction was revealed by Western-blot analysis. (G) Western blot analysis of cIAP1 in HeLa cells expressing wt cIAP1 or cIAP1Δα1, and treated for 1 hour with 1μM Birinapant (Birin.) or 17 nM GDC-0152 (GDC). HSC70 was used as a loading control. One representative experiment is shown.

Article Snippet: Lysates were pre-cleared using 20μl of Protein A/G+ Agarose beads (Sigma-Aldrich), and incubated overnight at 4°C with mouse IgG1 purified anti-HA.11 (Biolegend, Ozyme, Montigny-le-Bretonneux, France), goat polyclonal anti-cIAP1 antibody (R&D systems Europe, Lille, France) or mouse or goat IgG.

Techniques: Isolation, Produced, Bacteria, Incubation, Recombinant, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Activity Assay, Luciferase, Construct, Expressing

Journal: PLoS ONE

Article Title: E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

doi: 10.1371/journal.pone.0206253

Figure Lengend Snippet: (A) Cycloheximide chase experiment. Cells in which the indicated transgene expression was induced for 48 hrs were treated with cycloheximide 100μg/mL and E2F1 and cIAP1 expression were analyzed by Western blotting. The vector (◊) and cIAP1 (◆) control samples were loaded twice to compare the expression levels. β-actin was used as the loading control. E2F1 expression was quantified using Image J software (right panel). One representative experiment is shown. (B-D) Western blot analysis of 3HA-E2F1, cIAP1 myc-skp2 (B,D) or HA-cdh1 (C,D) in HeLa cells transfected with indicated constructs in the presence of control or cIAP1 siRNA. β-actin was used as the loading control. One representative experiment is shown (B,C). E2F1 and β-actin expression was quantified using Image J software (right panel). Mean ± S.D. of 3 independent experiments are shown (D). (E) cIAP1 modifies the ubiquitination profile of E2F1. HeLa cells were transfected with 3HA-E2F1, His-tagged ubiquitin wt or ubiquitin mutants containing only the K11, K48 or K63 constructs, in the presence of empty or cIAP1-encoding plasmids. E2F1 immunoprecipitated using anti-HA antibody and ubiquitinated was revealed using anti-poly-ubiquitin chain antibody (FK2). The expression of the constructs was checked by Western blot (lower panel).

Article Snippet: Lysates were pre-cleared using 20μl of Protein A/G+ Agarose beads (Sigma-Aldrich), and incubated overnight at 4°C with mouse IgG1 purified anti-HA.11 (Biolegend, Ozyme, Montigny-le-Bretonneux, France), goat polyclonal anti-cIAP1 antibody (R&D systems Europe, Lille, France) or mouse or goat IgG.

Techniques: Expressing, Western Blot, Plasmid Preparation, Software, Transfection, Construct, Immunoprecipitation

Journal: PLoS ONE

Article Title: E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

doi: 10.1371/journal.pone.0206253

Figure Lengend Snippet: ChIP-seq analysis of the recruitment of E2F1 onto chromatin in HeLa cells transfected with scrambled (Sc) or cIAP1-siRNA. Enriched DNA from ChIP and Input DNA fragments were end-repaired, extended with an ‘A’ base on the 3′end, ligated with indexed paired-end adaptors (NEXTflex, Bio Scientific) using the Bravo Platform (Agilent), size-selected after 4 cycles of PCR with AMPure XP beads (Beckman Coulter) and amplified by PCR for 10 more cycles (A). The efficiency of siRNA was checked by Western Blot analysis (B).

Article Snippet: Lysates were pre-cleared using 20μl of Protein A/G+ Agarose beads (Sigma-Aldrich), and incubated overnight at 4°C with mouse IgG1 purified anti-HA.11 (Biolegend, Ozyme, Montigny-le-Bretonneux, France), goat polyclonal anti-cIAP1 antibody (R&D systems Europe, Lille, France) or mouse or goat IgG.

Techniques: ChIP-sequencing, Transfection, Amplification, Western Blot

Journal: PLoS ONE

Article Title: E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

doi: 10.1371/journal.pone.0206253

Figure Lengend Snippet: GST-pull-down analysis of the interaction of E2F1 with cIAP1. GST-fusion proteins were produced in bacteria, immobilized on glutathione-sepharose and incubated in the presence of E2F1 recombinant protein (A) or HeLa cell lysate (B). The interaction was revealed by Western-blot analysis. Increasing concentrations of the Smac-N7 peptide (μg/mL) or the Smac mimetic BV6 (nM) or DMSO vehicle buffer corresponding to the higher concentration (0) were added to immobilized GST-fusion proteins 30 min before the interaction step (B). E2F1 and TRAF2 binding were quantified using Image J software and related to binding without IAC (lower panel). One representative experiment is shown.

Article Snippet: Blots were probed with the following antibodies: rabbit polyclonal anti-E2F1 (C20, Santa Cruz, Biotechnology, Clinisciences, Nanterre, France), goat polyclonal anti-GST (Rockland, Tebu-bio SAS, Le Perray en Yvelines, France), rabbit polyclonal anti-TRAF2 (Stressgen, Enzo Life Sciences, Villeurbanne, France), mouse monoclonal anti-HA.11 (Biolegend), mouse monoclonal anti-HSC70 (Santa Cruz Biotechnology), goat polyclonal anti-cIAP1 (AF8181, R&D systems Europe), mouse anti-Smac/DIABLO (BD Transduction Laboratories, BD Biosciences, Le Pont de Claix, France), mouse anti-Myc (Cell Signaling technology, Saint Quentin Yvelines, France), mouse anti-β-actin (AL978) and mouse anti-α-tubulin (clone AA13) (Sigma-Aldrich), monoclonal FK2-HRP-conjugated anti-ubiquitin (Enzo Life Sciences, Villeurbanne, France).

Techniques: Produced, Bacteria, Incubation, Recombinant, Western Blot, Concentration Assay, Binding Assay, Software

Journal: PLoS ONE

Article Title: E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

doi: 10.1371/journal.pone.0206253

Figure Lengend Snippet: (A) Primary structure of the cIAP1-BIR3 and BIR3 A-H deletion mutants. (B & C) GST-pull-down analysis of the interaction of E2F1 with the BIR3 deletion mutants described in A (B) or with wt cIAP1, isolated BIR3 or cIAP1 devoid of the BIR3 α1-helix (cIAP1Δα1) or the whole BIR3 (cIAP1ΔBIR3)(C). GST-fusion proteins were produced in bacteria, immobilized on glutathione-sepharose and incubated in the presence of E2F1 recombinant protein (B) or HeLa cell lysate (C). The interaction was revealed by Western-blot analysis. Mw: molecular weights. (D) Analysis of ubiquitination of E2F1 in HeLa cells transfected with wt 3HA-E2F1, His-Ubiquitin, an empty vector or a vector encoding wt cIAP1 or the BIR3-α1-helix deleted mutant. Ubiquitinated proteins were pulled-down using cobalt beads, and ubiquitinated E2F1 was revealed using an anti-E2F1 antibody. (E) E2F1 transcriptional activity was assessed in luciferase gene experiments using constructs in which luciferase expression was controlled by the CCNE or CAS7 promoter. Luciferase activity was normalized to β-galactosidase activity and expressed as fold-induction of promoter activity measured in cells transfected with empty vectors. The mean ± S.D. of at least 3 independent experiments is shown. Statistical analysis was performed using Student’s t test. (F) GST-pull-down analysis of the interaction of E2F1, Smac or TRAF2 with wt cIAP1, cIAP1 devoid of the BIR3 α1-helix (cIAP1Δα1) or cIAP1 altered in the TRAF2 interacting site (cIAP1-L47A). GST-fusion proteins were produced in bacteria, immobilized on glutathione-sepharose and incubated in the presence of HeLa cell lysate. The interaction was revealed by Western-blot analysis. (G) Western blot analysis of cIAP1 in HeLa cells expressing wt cIAP1 or cIAP1Δα1, and treated for 1 hour with 1μM Birinapant (Birin.) or 17 nM GDC-0152 (GDC). HSC70 was used as a loading control. One representative experiment is shown.

Article Snippet: Blots were probed with the following antibodies: rabbit polyclonal anti-E2F1 (C20, Santa Cruz, Biotechnology, Clinisciences, Nanterre, France), goat polyclonal anti-GST (Rockland, Tebu-bio SAS, Le Perray en Yvelines, France), rabbit polyclonal anti-TRAF2 (Stressgen, Enzo Life Sciences, Villeurbanne, France), mouse monoclonal anti-HA.11 (Biolegend), mouse monoclonal anti-HSC70 (Santa Cruz Biotechnology), goat polyclonal anti-cIAP1 (AF8181, R&D systems Europe), mouse anti-Smac/DIABLO (BD Transduction Laboratories, BD Biosciences, Le Pont de Claix, France), mouse anti-Myc (Cell Signaling technology, Saint Quentin Yvelines, France), mouse anti-β-actin (AL978) and mouse anti-α-tubulin (clone AA13) (Sigma-Aldrich), monoclonal FK2-HRP-conjugated anti-ubiquitin (Enzo Life Sciences, Villeurbanne, France).

Techniques: Isolation, Produced, Bacteria, Incubation, Recombinant, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Activity Assay, Luciferase, Construct, Expressing

Journal: PLoS ONE

Article Title: E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

doi: 10.1371/journal.pone.0206253

Figure Lengend Snippet: (A) Cycloheximide chase experiment. Cells in which the indicated transgene expression was induced for 48 hrs were treated with cycloheximide 100μg/mL and E2F1 and cIAP1 expression were analyzed by Western blotting. The vector (◊) and cIAP1 (◆) control samples were loaded twice to compare the expression levels. β-actin was used as the loading control. E2F1 expression was quantified using Image J software (right panel). One representative experiment is shown. (B-D) Western blot analysis of 3HA-E2F1, cIAP1 myc-skp2 (B,D) or HA-cdh1 (C,D) in HeLa cells transfected with indicated constructs in the presence of control or cIAP1 siRNA. β-actin was used as the loading control. One representative experiment is shown (B,C). E2F1 and β-actin expression was quantified using Image J software (right panel). Mean ± S.D. of 3 independent experiments are shown (D). (E) cIAP1 modifies the ubiquitination profile of E2F1. HeLa cells were transfected with 3HA-E2F1, His-tagged ubiquitin wt or ubiquitin mutants containing only the K11, K48 or K63 constructs, in the presence of empty or cIAP1-encoding plasmids. E2F1 immunoprecipitated using anti-HA antibody and ubiquitinated was revealed using anti-poly-ubiquitin chain antibody (FK2). The expression of the constructs was checked by Western blot (lower panel).

Article Snippet: Blots were probed with the following antibodies: rabbit polyclonal anti-E2F1 (C20, Santa Cruz, Biotechnology, Clinisciences, Nanterre, France), goat polyclonal anti-GST (Rockland, Tebu-bio SAS, Le Perray en Yvelines, France), rabbit polyclonal anti-TRAF2 (Stressgen, Enzo Life Sciences, Villeurbanne, France), mouse monoclonal anti-HA.11 (Biolegend), mouse monoclonal anti-HSC70 (Santa Cruz Biotechnology), goat polyclonal anti-cIAP1 (AF8181, R&D systems Europe), mouse anti-Smac/DIABLO (BD Transduction Laboratories, BD Biosciences, Le Pont de Claix, France), mouse anti-Myc (Cell Signaling technology, Saint Quentin Yvelines, France), mouse anti-β-actin (AL978) and mouse anti-α-tubulin (clone AA13) (Sigma-Aldrich), monoclonal FK2-HRP-conjugated anti-ubiquitin (Enzo Life Sciences, Villeurbanne, France).

Techniques: Expressing, Western Blot, Plasmid Preparation, Software, Transfection, Construct, Immunoprecipitation

Journal: PLoS ONE

Article Title: E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding

doi: 10.1371/journal.pone.0206253

Figure Lengend Snippet: ChIP-seq analysis of the recruitment of E2F1 onto chromatin in HeLa cells transfected with scrambled (Sc) or cIAP1-siRNA. Enriched DNA from ChIP and Input DNA fragments were end-repaired, extended with an ‘A’ base on the 3′end, ligated with indexed paired-end adaptors (NEXTflex, Bio Scientific) using the Bravo Platform (Agilent), size-selected after 4 cycles of PCR with AMPure XP beads (Beckman Coulter) and amplified by PCR for 10 more cycles (A). The efficiency of siRNA was checked by Western Blot analysis (B).

Article Snippet: Blots were probed with the following antibodies: rabbit polyclonal anti-E2F1 (C20, Santa Cruz, Biotechnology, Clinisciences, Nanterre, France), goat polyclonal anti-GST (Rockland, Tebu-bio SAS, Le Perray en Yvelines, France), rabbit polyclonal anti-TRAF2 (Stressgen, Enzo Life Sciences, Villeurbanne, France), mouse monoclonal anti-HA.11 (Biolegend), mouse monoclonal anti-HSC70 (Santa Cruz Biotechnology), goat polyclonal anti-cIAP1 (AF8181, R&D systems Europe), mouse anti-Smac/DIABLO (BD Transduction Laboratories, BD Biosciences, Le Pont de Claix, France), mouse anti-Myc (Cell Signaling technology, Saint Quentin Yvelines, France), mouse anti-β-actin (AL978) and mouse anti-α-tubulin (clone AA13) (Sigma-Aldrich), monoclonal FK2-HRP-conjugated anti-ubiquitin (Enzo Life Sciences, Villeurbanne, France).

Techniques: ChIP-sequencing, Transfection, Amplification, Western Blot