FIG. 6.

The level of SUMOylated SF-1 is not altered by ACTH treatment. (A) Y1 cells (1 × 10⁶) were seeded in 10-cm plates and 24 h later were serum deprived in DMEM supplemented with 0.05% bovine serum albumin, followed by transfection with 3 μg HA-SUMO3 expression vector alone (−) or together with the indicated SF-1 or mutant SF-1 expression vector. Twenty-four hours after transfection, the cells were treated with 2.5 μM α-amanitin for 2 h. The cells were washed twice with PBS, and fresh serum-free medium was added 30 min prior to ACTH (10 nM) stimulation for the indicated times. The cells were harvested, and the lysates were subjected to Ni²⁺ bead pulldown, followed by anti-FLAG (top) or anti-HA (bottom) immunoblotting. The empty arrowhead indicates SUMOylated SF-1. The solid arrowhead indicates non-SUMOylated SF-1. (B) Lysates of Y1 cells (1 × 10⁶) stably expressing HIS-FLAG WT-SF-1 and incubated for the indicated times with ACTH were subjected to Ni²⁺ bead pulldown, followed by anti-SF-1 immunoblotting (IB).