Nitrocellulose membranes

Western blot analysis was performed as described previously (26 (link)). Briefly, the cells were lysed in RIPA lysis buffer (Sigma-Aldrich; Merck KGaA) and protein concentration was measured using a Pierce™ Rapid Gold BCA Protein assay kit (Thermo Fisher Scientific, Inc.). Subsequently, 20 µg protein/lane was separated via 4–20% SDS-PAGE. The proteins were then transferred to nitrocellulose membranes (Qiagen GmbH) and after blocking with 5% BSA (Beyotime Institute of Biotechnology) at 37°C for 1.5 h, the membranes were incubated with the following specific primary antibodies overnight at 4°C: Rabbit monoclonal anti-SIRT2 (1:2,000; Abcam; cat. no. ab211033), rabbit polyclonal anti-HRAS (1:2,000; ProteinTech Group, Inc.; cat. no. 15531-1-AP), rabbit monoclonal anti-ERK1/2 (1:10,000; Abcam; cat. no. ab109282), rabbit monoclonal anti-p-ERK1/2 (1:10,000; Abcam; cat. no. ab223500), rabbit polyclonal anti-PI3K (1:1,000; Abcam; cat. no. ab154598), rabbit polyclonal anti-p-PI3K (1:1,000; Abcam; cat. no. ab182651) and anti-GAPDH (1:10,000; Abcam; cat. no. ab245355). The following day, the membranes were incubated with goat anti-rabbit IgG H&L (HRP) (1:20,000; Abcam; cat. no. ab97047) at 37°C for 1.5 h. The EasyBlot ECL kit (Sangon Biotech Co., Ltd.) was used for chemiluminescence detection. GAPDH was used as a loading control.