Immunoblot Analyses of mTORC1/2 Pathway Activity.

Mouse ovaries (n = 30) were placed in liquid nitrogen at the time of sacrifice. A minimum of three ovaries from each treatment group were individually homogenized in a buffer of 150 mM NaCl, 50 mM Tris⋅HCl (pH 8), 1 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 25 mM β-glycerophosphate, 2 mM Na₃VO₄, and Protease Inhibitor (EDTA-free) tablets (Thermo Fisher Scientific). Protein concentration was determined by the bicinchoninic acid assay. To determine the total levels and phosphorylation status of specific proteins, equal amounts of protein were resolved by SDS/PAGE in 8%, 10%, or 12% gels as indicated and transferred to 0.45-μm PVDF membranes (Millipore) for immunoblotting. The following Cell Signaling antibodies were used: mTOR [rabbit monoclonal antibody (mAb) 2972], S2448 phospho-mTOR (rabbit mAb 2971), AKT (rabbit mAb 9272), S473 phospho-AKT (rabbit mAb 9271), S6 (rabbit mAb 2217), phospho-S6 (rabbit mAb 2211), 4E-BP1 (rabbit mAb 9452), S65 phospho-4E-BP1 (rabbit mAb 9451), and anti-rabbit eEF2 or eIF4A to verify equal loading, all used at 1:1,000 dilution. The Enhanced Chemiluminescence (ECL; GE Healthcare) or SuperSignal (Thermo Scientific) procedure was used to visualize protein signals as described by the manufacturers. The light intensity of each band was quantified with ImageJ software (NIH).