LRH-1 is modified by SUMO-1 in vivo. (A) Extracts of HEK-293 cells transfected with the indicated expression vectors were analyzed in Western blots with a myc tag-specific antibody to detect modified and unmodified LRH-1. (B) myc-LRH-1 and HA-SUMO-1 proteins were immunoprecipitated with αmyc or αHA antibody, and the presence of LRH-1 and SUMO-1 in the immunoprecipitates was detected in Western blots by using αLRH-1, αmyc and αHA antibodies as indicated. (C) Endogenous LRH-1 from C3A-HepG2 cells was detected in Western blot by an αLRH-1 antibody (left panel) or immunoprecipitated with αLRH-1 antibody and analyzed in Western blots with αSUMO-1 antibody. As a control, nonimmune (Non-Imm.) antiserum was used for immunoprecipitation. (D) C3A-HepG2 cells were transfected with SUMO siRNA and control siRNA as indicated. Cell lysates prepared 72 h after transfection were analyzed in Western blots with antibodies recognizing SUMO-1, HNF4, TFIIB, and LRH-1 (left and middle panels). Part of the extracts was immunoprecipitated with αLRH-1 antibody and analyzed in Western blots with αSUMO-1 antibody (panel at right).