Anti ach4

Western blot analysis was performed in accordance with the standard protocol. Total cell lysate was prepared using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) with protease inhibitor cocktail. Proteins 20 µg were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrotransferred to nitrocellulose blotting membranes (0.2 µm; GE Healthcare Life Science, Uppsala, Sweden). Membranes were incubated with 5% non-fat milk for three hours and incubated with primary antibodies overnight at 4°C. Fluorescence-conjugated secondary antibody (goat anti-rabbit/mouse IRDye; LI-COR Biosciences, Lincoln, NE, USA) diluted in the blocking buffer (1:5000) was incubated for one hour at RT, and protein bands were detected using Odyssey infrared imaging system (LI-COR Biosciences). Primary antibodies were as follows: anti-SIRT1 (1:1000, CST), anti-Cortactin (1:1000; CST), anti-Acetylated-lysine (1:1000; CST), anti-Ac-Cortactin (1:1000; Millipore), anti-Histone3 (H3; 1:1000; CST) anti-Ac-H3 (1:1000; Millipore), anti-Histone4 (H4; 1:1000; CST), anti-Ac-H4 (1:1000; Millipore), anti-β-Actin (1:1000; CST). The protein bands were analyzed by Image J (NIH) software.