Fig. 8.

Mutations in the LBP that Reduce Transcriptional Activity Is Less Phosphorylated by CDKs

COS-1 cells were transfected with pCDNA3.1+/SF1 (WT; 50 ng) or LBP mutants (pCDNA3.1Zeo+/SF1-A270W, L348W, L345W, V349W, and A434W; 50 ng) together with the reporter gene construct CYP17/Luc (0.5 μg) (panel A) or MC2R/Luc (panel B). The luciferase activities are presented as fold induction compared with the activity in the absence of SF1 and relatively to the activities from pCMV5/βgal. The experiments were repeated three times, shown as means ± sds (CYP17/Luc, n = 9; *, P < 0.05; ***, P < 0.001; as determined by one-way ANOVA, Bonferoni posttest; MC2R/Luc, n = 6; *, P < 0.05, as determined by nonparametric ANOVA, Dunn posttest). C, COS-1 cells were transfected with SF1 (WT) or LBP mutants (5 μg), and SF1 was isolated by DPD. The isolated proteins were subjected to Western blotting analyses using anti-P-Sub-CDK (upper panel) and SF1 antibodies (lower panel). Densitometric analyses were performed and are shown as ratios between phosphorylated SF1 and total SF1. ACTHR, ACTH receptor.