sfSGK is activated by PDK1 and TORC2 in a PI3K-dependent manner. (A) Unstimulated oocytes were treated with 1-MA and analyzed by immunoblotting with anti-human phospho-SGK (sfSGK-pT312 [A-loop]), anti-sfSGK-HM (sfSGK), anti-sfAkt phospho-Ser477 (Akt-pS477[HM]), anti-sfAkt C-terminal fragment (Akt), anti-sfCdc25 (Cdc25), anti-Cdk1 phospho-Tyr15 (Cdk1-pY15), and anti-PSTAIR (Cdk1) antibodies. GVBD occurred at 17 min. (B) A piece of ovary was separated into oocytes and germinal epithelium, and subjected to immunoblotting. (C) To stimulate ovarian oocytes, 1-MA was injected into the body cavity of female starfish. Pieces of ovaries were then recovered at the indicated times and analyzed via immunoblotting or monitored for GVBD timing. GVBD occurred within 25 min. Spawning started at 30 min. (D) Unstimulated oocytes were treated with 1-MA for 4 min in the presence of indicated inhibitors or DMSO, and then subjected to normal SDS-PAGE, followed by immunoblotting. Closed and open arrowheads in A–D indicate the positions of the upper and lower sfSGK bands, respectively. Asterisks in A–D, nonspecific bands. (E and F) Unstimulated oocytes were treated with 1-MA for 4 min (E) or treated as in D (F), and then subjected to Phos-tag SDS-PAGE, followed by immunoblotting (IB). The results shown are representative of three independent experiments in A, E, and F, and two independent experiments in B–D.