P mtor

Total proteins in placenta tissue or cells were extracted with RIPPA lysis buffer (Beyotime, China) and quantified the concentration by BCA Protein Assay Kit (Beyotime, China). Protein samples were separated using 10% or 12% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, Canada). Membranes were blocked with 5% milk (Boster, China), incubated overnight at 4 °C with primary antibody and further incubated with HRP-labelled corresponding source of secondary antibody (Boster, China) at 37 °C for 1 h. Primary antibodies (Zhongshan, China: ACTB, CST: LC3, #83,506, Atg5, #12,994, Beclin-1, #3495, P62, #5114, mTOR, #2983, p-mTOR, #5536, Akt, #4691, p-Akt, #4060, Raptor, #2280, p-Raptor, #2083, AMPK, #5832, p-AMPK, #2535, Santa: PINK1, sc-517353, Parkin, sc-32282, Phb2, sc-133094) were diluted in 5% milk in PBST. The band intensity was visualized using enhanced chemiluminescence reagent (Bio-Rad, USA) and quantified by Image J software.

Equal amounts of each protein sample was loaded to detect protein expression. The following primary antibodies were used in this experiment: β-actin (Beyotime, AA128, Shanghai, China), ELF2 (Proteintech, 12499-1-AP, United States), Bax (BBI, D220073, Shanghai, China), Bcl-2 (BBI, D260117, Shanghai, China), PI3K p110 beta (Bioss, bs-6423R, Beijing, China), p-PI3K p110 beta (Bioss, bs-6417R, Beijing, China), AKT (Cell Signaling Technology, 4685, United States), p-AKT (Cell Signaling Technology, 4060, United States), mTOR (Boster Biological Technology, BM4182, United States), and p-mTOR (Boster Biological Technology, BM4840, United States). Horse Radish Peroxidase-conjugated goat anti-rabbit IgG secondary antibodies were purchased from Beyotime (Shanghai, China).

The proteins of GMEC were harvested using RIPA lysis buffer with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). Protein quantification was performed using the BCA Protein Assay Kit (Solarbio, Beijing, China), and the same amount of protein was heated with SDS-PAGE loading buffer (Solarbio, Beijing, China) at 98 °C for 10 min to detect specific protein expression. Antibodies specific for MAPK (Cell Signaling Technology, Danvers, MA, 9102, USA), phosphorylated-MAPK (Cell Signaling Technology, Danvers, MA, 4376, USA), mTOR (Boster Biological Technology, Pleasanton, CA, BM4182, USA), p-mTOR (Boster Biological Technology, Pleasanton, CA, BM4840, USA), STAT5 (Cell Signaling Technology, Danvers, MA, 94205, USA), p-STAT5 (Cell Signaling Technology, Danvers, MA, 9359, USA), STC1 (Boster, ba2983-2, Wuhan, China), MMP1 (BBI, D220093, Shanghai, China), and β-actin (Beyotime, AA128, Shanghai, China) were applied.