Fig. 4
RARγ C fragment are important for protecting cell from TNF-induced necroptosis via interaction with RIP1. a Immunoprecipitation of HT-29 cells treated with TSZ for the indicated times. Cell lysates were immunoprecipitated with anti-RARγ antibody and analyzed by immunoblotting with the indicated antibodies. b HEK293T cells were co-transfected with wild-type V5-RARγ (WT) or the V5-RARγ-NLSmut (NLS) and with or without FLAG-RIP1 plasmids. Cell lysates were immunoprecipitated using anti-FLAG (RIP1) antibody and analyzed with the indicated antibodies. c Scheme of RARγ and RIP1 genes (upper panel). HEK293T cells were co-transfected with different fragment of RARγ-V5 (F: full; N:1–201aa; C: 188–454aa) and with or without FLAG-RIP1 plasmids; or with different fragment of His-Xpress-RIP1 (F: full; N: 1–324aa; C: 324–671aa; DD: 560–669aa) and with or without RARγ-V5. Cell lysates were immunoprecipitated using anti-FLAG (RIP1) or anti-V5 (RARγ) antibody and analyzed with the indicated antibodies. d HT-29 RARγ-shRNA-A cells infected with rRARγ-C. Cell death analysis of HT-29 cont-shRNA, RARγ-shRNA-A, and RARγ-shRNA-A + rRARγ-C when treated with TSZ for 24 h was determined by PI staining using flow cytometry (upper left panel). (*P < 0.05 versus cont-shRNA; # P < 0.05 versus RARγ-shRNA-A; ANOVA). The bars represent the mean ± s.e.m. of three experiments. Western blot analysis of cells as mentioned in left panel (upper right panel). Confocal microscopy of HT-29 cells infected with GFP-RARγ-C plasmid (lower left panel) (blue: DAPI; green: RARγ). Western blot analysis of HEK293T cells were transfected with p-EGFP, p-EGFP-RARγ and p-EGFP-RARγ-C by using anti-GFP and anti-Actin antibodies (lower right panel). All blots and images above are representative of one of three experiments
