Journal: Nature Communications

Article Title: The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited

doi: 10.1038/s41467-017-00496-6

Figure Lengend Snippet: RARγ has a role in the formation of death complexes. a Western blot analysis of HT-29 cont-shRNA, and RARγ-shRNA-A treated with DMSO, TS or TSZ for 24 h and cell lysates were immunoblotted with the indicated antibodies. b , c Immunoprecipitation of HT-29 cont-shRNA or RARγ-shRNA-A cells treated with TSZ for the indicated time. Cell lysates were collected and immunoprecipitated with anti-Caspase-8 antibody b or anti-TNFR1 antibody c . The immunoprecipitated complexes were immunoblotted with the indicated antibodies. d Sequential immunoprecipitation of HT-29 cont-shRNA or RARγ-shRNA-A cells treated TSZ for 2 h. First IP : TNFR1 complex I was immunoprecipitated using anti-TNFR1 antibody. Second IP : the remaining lysates were immunoprecipitated again with anti-TNFR1 antibody. Third IP : the remaining lysates were then immunoprecipitated with anti-TRADD antibody. The immunoprecipitated complexes were analyzed by with the indicated antibodies. (M: marker). All blots above are representative of one of three experiments

Article Snippet: Anti-RARγ (C-15) (sc-550) for human, anti-RARα (C-20) (sc-551), anti-caspase-8 (C-20) (sc-6136), anti-cIAP2 (sc7944) and anti-Fas (C-20) (sc-715) from Santa Cruz; anti-RIP1 (610459) and anti-FADD (610400) from BD Biosciences; anti-RARγ1 (ab5905) for mouse, anti-RIP3 (ab72106), anti-MLKL (ab184718) for human, anti-MLKL (ab172868) for mouse and anti-cIAP1 (ab2399) from Abcam; anti-RIP3 (2283) for mouse from ProSci, anti-TRADD (05-473) from Upstate; anti-TRAF2 (MAB3277) and anti-TNFR1 (AF-425-PB) from R&D; anti-Actin (A3853) (dilution 1:10,000), anti-FLAG (F9291) (dilution 1:5,000) and anti-GFP (G6539) (dilution 1:5 000) from Sigma; anti-V5 (R960-25) (dilution 1:5,000) from Invitrogen; anti-PARP1 (BML-SA250-0050) from Enzo Life Science; anti-GAPDH (NB300-22) (dilution 1:5,000) from Novus Biologicals; anti-cleaved caspase-8 (9496), anti-CYLD (4495), anti-RIP1 (137451) and anti-p-RIP1(65746) from Cell Signaling Technology; anti-DsRed (632392) (dilution 1:5,000) from Clontech.

Techniques: Western Blot, shRNA, Immunoprecipitation, Marker

Journal: Nature Communications

Article Title: The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited

doi: 10.1038/s41467-017-00496-6

Figure Lengend Snippet: RARγ initiates the formation of death complexes by dissociating RIP1 from TNFR1. a Immunoprecipitation of HT-29 cont-shRNA, TRADD-shRNA or RIP1-shRNA treated with TSZ for the indicated times. Cell lysates were immunoprecipitated using anti-TNFR1 antibody and immunoblotted with the indicated antibodies. b Sequential immunoprecipitation of HEK293T cells co-transfected with FLAG-TNFR1, DsRed-RIP1 and increasing amounts of V5-RARγ-NLSmut plasmids as indicated. First IP : cell lysates were immunoprecipitated with anti-FLAG (TNFR1) antibody. Second IP : the remaining lysates were then immunoprecipitated with anti-V5 (RARγ) antibody. The immunoprecipitated complexes were analyzed by with the indicated antibodies. c Sequential immunoprecipitation of HEK293T cells co-transfected with V5-RARγ-NLSmut, RIP1-Myc, and increasing amounts of DsRed-RIP3 plasmids as indicated. First IP : cell lysates were immunoprecipitated with anti-V5 (RARγ) antibody. Second IP : the remaining lysates were then immunoprecipitated with anti-DsRed (RIP3) antibody. The immunoprecipitated complexes were analyzed with the indicated antibodies. d WT and RIP3−/− MEFs treated with TSZ for the indicated times. Cell lysates were immunoprecipitated using anti-Caspase-8 antibody and immunoblotted with the indicated antibodies. All blots above are representative of one of three experiments

Article Snippet: Anti-RARγ (C-15) (sc-550) for human, anti-RARα (C-20) (sc-551), anti-caspase-8 (C-20) (sc-6136), anti-cIAP2 (sc7944) and anti-Fas (C-20) (sc-715) from Santa Cruz; anti-RIP1 (610459) and anti-FADD (610400) from BD Biosciences; anti-RARγ1 (ab5905) for mouse, anti-RIP3 (ab72106), anti-MLKL (ab184718) for human, anti-MLKL (ab172868) for mouse and anti-cIAP1 (ab2399) from Abcam; anti-RIP3 (2283) for mouse from ProSci, anti-TRADD (05-473) from Upstate; anti-TRAF2 (MAB3277) and anti-TNFR1 (AF-425-PB) from R&D; anti-Actin (A3853) (dilution 1:10,000), anti-FLAG (F9291) (dilution 1:5,000) and anti-GFP (G6539) (dilution 1:5 000) from Sigma; anti-V5 (R960-25) (dilution 1:5,000) from Invitrogen; anti-PARP1 (BML-SA250-0050) from Enzo Life Science; anti-GAPDH (NB300-22) (dilution 1:5,000) from Novus Biologicals; anti-cleaved caspase-8 (9496), anti-CYLD (4495), anti-RIP1 (137451) and anti-p-RIP1(65746) from Cell Signaling Technology; anti-DsRed (632392) (dilution 1:5,000) from Clontech.

Techniques: Immunoprecipitation, shRNA, Transfection

Journal: Nature Communications

Article Title: The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited

doi: 10.1038/s41467-017-00496-6

Figure Lengend Snippet: RARγ modulates TNF-induced RIP-initiated cell death. a TRADD is responsible for initiated TC or TCZ-induced cell death. b RARγ is essential for the transition from inflammatory signaling to death pathways in TNF-induced RIP1-initiated cell death. When RARγ is released from the nucleus in response to TNF under the conditions of cIAP inhibition, it initiates the formation of death complex IIa by dissociating RIP1 from TNFR1. When RIP1 recruits RIP3 to necrosome/complex IIb, RARγ is replaced from the complex

Article Snippet: Anti-RARγ (C-15) (sc-550) for human, anti-RARα (C-20) (sc-551), anti-caspase-8 (C-20) (sc-6136), anti-cIAP2 (sc7944) and anti-Fas (C-20) (sc-715) from Santa Cruz; anti-RIP1 (610459) and anti-FADD (610400) from BD Biosciences; anti-RARγ1 (ab5905) for mouse, anti-RIP3 (ab72106), anti-MLKL (ab184718) for human, anti-MLKL (ab172868) for mouse and anti-cIAP1 (ab2399) from Abcam; anti-RIP3 (2283) for mouse from ProSci, anti-TRADD (05-473) from Upstate; anti-TRAF2 (MAB3277) and anti-TNFR1 (AF-425-PB) from R&D; anti-Actin (A3853) (dilution 1:10,000), anti-FLAG (F9291) (dilution 1:5,000) and anti-GFP (G6539) (dilution 1:5 000) from Sigma; anti-V5 (R960-25) (dilution 1:5,000) from Invitrogen; anti-PARP1 (BML-SA250-0050) from Enzo Life Science; anti-GAPDH (NB300-22) (dilution 1:5,000) from Novus Biologicals; anti-cleaved caspase-8 (9496), anti-CYLD (4495), anti-RIP1 (137451) and anti-p-RIP1(65746) from Cell Signaling Technology; anti-DsRed (632392) (dilution 1:5,000) from Clontech.

Techniques: Inhibition