Fig. 3

PIAS1 promotes PPARγ SUMOylation by its SUMO E3 ligase activity. a 293 T cells were cotransfected with Flag-PPARγ and HA-SUMO1 plasmids as indicated. The cell lysates were immunoprecipitated (IP) with anti-Flag antibody, followed by blotting (IB) with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted (IB) with anti-HA antibody. b H9C2 cell lysates were immunoprecipitated with anti-PPARγ antibody, followed by blotting with anti-SUMO1 or anti-PPARγ antibody. c 293 T cells were cotransfected with vector, Flag-PPARγ and HA-tagged SUMO E3 ligases plasmids as indicated. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by blotting with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted with anti-HA antibody. d 293 T cells were cotransfected with Flag-PPARγ, HA-SUMO1 and siRNAs against selected SUMO E3 ligases as indicated (control cells were transfected without HA-SUMO1). The cell lysates were immunoprecipitated with anti-Flag antibody, followed by blotting with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted with anti-HA antibody. e Quantification of the densitometry of the SUMO-PPARγ band in D (n = 3). f 293 T cells were cotransfected with Flag-PPARγ, HA-SUMO1 and siRNAs against PIAS1 as indicated (control cells were transfected without HA-SUMO1). The cell lysates were immunoprecipitated with anti-Flag antibody, followed by blotting with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted with anti-PIAS1 antibody. g Quantification of the densitometry of the SUMO-PPARγ band in f (n = 3). h H9C2 cells were transfected with siRNAs against PIAS1. Cell lysates were immunoprecipitated with anti-PPARγ antibody, followed by blotting with anti-SUMO1 or anti-PPARγ antibody. Whole-cell lysates were blotted with anti-SUMO1 antibody. i 293 T cells were cotransfected with Flag-PPARγ, HA-SUMO1 and increasing doses of PIAS1 wild-type (PIAS1-WT) or PIAS1 catalytic mutant (PIAS1-Mut) as indicated. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by blotting with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted with anti-PIAS1 antibody. j Quantification of the densitometry of the SUMO-PPARγ band in I (n = 3). All these experiments were performed three times. Data presented are means ± SD, ^***P < 0.001 vs. mock group, ^**P < 0.01 vs. scramble group, and ^##P < 0.01 vs. control group