Anti ps6k1 antibody

Cultured cells or heart tissues were used to extract total protein with RIPA lysis buffer (Millipore) with protease inhibitor cocktail (Biomake). Next, standard Western blot was performed to determine the levels of interested proteins according to a protocol modified from previous studies [31 (link), 32 (link)]. The primary antibodies used for Western blot assay are :
Anti-IDO1 antibody (Abcam; 1:1000), anti-GAPDH antibody (Proteintech; 1:2000), anti-pAKT antibody (Cell Signaling Technology; 1:1000), anti-AKT antibody (Cell Signaling Technology; 1:1000), anti-pmTOR antibody (Cell Signaling Technology; 1:1000), anti-mTOR antibody (Cell Signaling Technology; 1:1000), anti-pS6K1 antibody (Cell Signaling Technology; 1:1000), anti-S6K1 antibody (Cell Signaling Technology; 1:1000). The secondary antibodies (1:5000) and ECL kit were purchased from Servicebio.

Total proteins were extracted from brain tissues and neurons with RIPA buffer (Beyotime, P0013) supplied with proteinase inhibitor cocktail (Roche, 04693124001) and phosphatase inhibitor cocktail (Sigma, P5726). Total protein (30 μg) was subjected to SDS-PAGE for protein separation. The proteins were transferred to PVDF membranes and blocked with 5% fat-free milk in TBST buffer, and then the membranes were incubated with individual primary antibodies overnight. Then the membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshanjinqiao) for 2 h. Finally, the secondary antibodies were detected by SuperSignal™ Chemiluminescent HRP Substrates (Thermo Fisher, 32106). The following primary antibodies were used in the present study: anti-GAPDH antibody (Santa Cruz, sc-47724), anti-BCAT1 antibody (Novus Biological, NBP2-01826), anti-Tau antibody (Abcam, ab64193), anti-p-Tau antibody (Abcam, ab109390), anti-mTOR antibody (Cell Signaling Technology, 2983), anti-p-mTOR antibody (Cell Signaling Technology, 5536), anti-S6K1 antibody (Cell Signaling Technology, 9202), and anti-p-S6K1 antibody (Cell Signaling Technology, 9206).