FIG. 1.

SF-1 SUMOylation. (A) Sequence alignment of the human, mouse, and pig SF-1 proteins showing the regions that contain the two potential SUMO sites (K119 and K194) and the phosphorylation site (S203). (B) COS-7 cells (2 × 10⁶) were seeded in 10-cm plates and transfected 24 h later with 5 μg of SF-1 receptor and HA-SUMO3 expression vectors as indicated. After 48 h, cells were harvested and subjected to an in vivo SUMOylation assay as described in Materials and Methods. The cell lysates were subjected to Ni²⁺ bead pulldown, followed by anti-FLAG (left) or anti-HA (right) immunoblotting. The empty arrowheads indicate SUMOylated SF-1. The solid arrowhead indicates non-SUMOylated SF-1. (C) Schematic representation of the mouse SF-1 protein with the lysine-to-arginine and serine-to-alanine/aspartate SF-1 mutants generated in this study to determine potential SUMOylation and phosphorylation sites on SF-1. (D) Expression of HIS-FLAG-tagged SF-1 in stable Y1 cell lines. Lysates of Y1 cells (1 × 10⁶) expressing HIS-FLAG-tagged WT SF-1 (WT), SUMO mutant SF-1 (K119R, K194R, and 2KR), phosphomutant SF-1 (S203A and S203D), or combined SUMO and phosphomutant SF-1 (K194RS203A and K194RS203D) were subjected to either anti-SF-1 immunoblotting (top) or Ni²⁺ bead pulldown, followed by anti-SF-1 immunoblotting (middle) or by anti-FLAG immunoblotting (bottom).