Ab66138

Cultures were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Primary antibodies against NFH (Abcam, ab72996; 1∶1000), synapsin-I (Millipore, AB1543P; 1∶100), activated caspase-3 (BioVision, 3015–100; 1∶100) and phosphorylated (S473) Akt (Abcam ab66138; 1∶200) were diluted in blocking solution (5% donkey serum, 1 mg/ml BSA IgG and protease free, in PBS) and incubated overnight at 4°C. Samples were incubated with species-specific secondary antibodies (Jackson Immunoresearch) for 2 h at room temperature. TMR-conjugated α-bungarotoxin (Sigma, T0195) staining for evidence of acetylcholine receptor (AChR) clusters was performed at 1 µg/ml in PBS for 15 min prior to cell permeabilization and primary antibody incubation. For visualization of nuclei in myotubes, Hoechst 33258 (1 µg/ml) or DAPI was used for live or fixed samples, respectively. After the staining protocol was completed, MFC was peeled from the dish by gently pulling it from the proximal to distal side, to minimize severing of axons. Prolong mounting medium was added and covered with a #1.5, 18×18 mm coverslide.

Proteins were extracted from ovaries tissues with RIPA (Beyotime, P0013C) lysis buffer for 30 min on ice with frequent vortexing. SDS-PAGE sample loading buffer was added into the lysate which was subsequently boiled for 5 min. Then lysates were collected by centrifugation at 14,000 rpm for 5 min. Total proteins were separated by SDS-PAGE with a 5% stacking gel and a 10% separating gel for 50 min at 100 V and 2.5 h at 120 V, respectively, and then electrophoretically transferred onto PVDF membrane. After blocked at room temperature in TBST buffer containing 10% BSA (Sigma), the membranes were hybridized with primary antibodies overnight at 4°C. Finally, the membranes were washed with TBST for three times, then incubated for 2 h at room temperature with secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Beyotime, A0208) at a dilution of 1:1000 in TBST. Membranes were washed three times for 5 min each in TBST. Signals were analyzed by the enhanced chemiluminescence (ECL) detection system. The beta Actin antibodies were detected as a control. Primary antibodies including rabbit anti-AKT polyclonal antibody (Abcam, ab86926), rabbit anti-AKT (phospho S473) polyclonal antibody (Abcam, ab66138), rabbit anti-beta Actin polyclonal antibody (Abcam, ab8227) were used in this study.

Protein was extracted from livers using RIPA lysis buffer containing protease/phosphatase inhibitor cocktail (Beyotime Biotechnology). Antibodies against p-AKT (phospho Ser473, ab66138, Abcam), AKT (#4691, Cell Signaling Technology), p-GSK3β (phospho Ser9, #9322, Cell Signaling Technology), and GSK3β (#12456, Cell Signaling Technology) were used.