Figure 5. TA inhibits exogenous and endogenous NR5A sumoylation, as well as general sumoylation in cells.

(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

Figure 5—figure supplement 1. UBC9 transcripts and protein levels following siUBC9 knockdown in HepG2 hLRH-1 cells.

(A) Relative expression of UBC9 (left panel) and hLRH-1 (right panel) in HepG2 cells following 72 hr exposure to siControl (siCont) or siUBC9 as described in ‘Materials and methods’. (B) Immunoblot of UBC9 protein following 72 hr exposure to siCont or siUBC9 with percentage decrease of UBC9 protein shown in bar graph (right panel).

DOI: http://dx.doi.org/10.7554/eLife.09003.016

Figure 5—figure supplement 2. TA attenuates endogenous RanGap sumoylation.

Endogenous sumoylated (Su-RanGap 1x) and unmodified RanGap (RanGap) in HepG2 hLRH-1 cells after siCont, siUBC9, or after TA treatment (6 hr) with concentrations indicated. Note that the anti-RanGap rabbit monoclonal antibody from GeneTex recognizes both 1x Sumoylated RanGap and unmodified RanGap.

DOI: http://dx.doi.org/10.7554/eLife.09003.017