(A) Levels of unsumoylated (LRH-1) and sumoylated hLRH-1 species in HepG2 cells detected with anti-Flag following TA, 2-D08, or GA at specified concentrations after 24 hr treatment. Vehicle control (DMSO) is shown (-). (B) Cell viability for HepG2 cells expressing wild-type hLRH-1 following 24 hr treatment with increasing concentrations of GA or TA. (C) Sumoylation of wild-type hLRH-1 expressed in HepG2 cells and detected with anti-Flag after siCont or siUBC9 knockdown for 72 hr (+), and with increasing TA (6 h). Shorter exposure of 1x-sumoylated hLRH-1 is shown in panel below (1x), as well as loading control (GAPDH). Vehicle control (DMSO) is shown (-). (D) Flag-tagged hLRH-1 protein immunoprecipitated by anti-Flag in HepG2 cells treated with vehicle or TA (30 or 50 μM) followed by immunoblot with either anti-SUMO1 (top panel) or anti-SUMO2 (bottom panel). Arrows indicated migration of 1x, 2x, and 3x sumoylated hLRH-1 species. (E) Levels of total sumoylated or ubiquitinated proteins in HepG2 cells following siControl and siUBC9 (72 hr) or TA treatment (6 hr), as detected by anti-SUMO1, -SUMO2, or -ubiquitin. (F) Effects of TA (6 hr) on endogenous SF-1 sumoylation in H295R cells and detected anti-SF-1 antibody. GA: Ginkgolic acid; TA: Tannic acid.
DOI:
http://dx.doi.org/10.7554/eLife.09003.015