Peroxidase labeled anti rabbit or anti mouse secondary antibodies

The expression of protein in cells was evaluated by Western blotting. Whole cell lysates (WCL) were prepared by extraction in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA), followed by protein quantification using a bicinchoninic acid assay (Applygen, Beijing, China) and lysis in Laemmli sample buffer. A total of 30 μg of the protein sample was run on a 10% Tris‐glycine gel and transferred to nitrocellulose. Primary antibodies were added and incubated overnight at 4°C, and secondary antibodies were conjugated to horseradish peroxidase for two hours at room temperature. Blots were developed by enhanced chemiluminescence and photographed using Fujifilm Dark Box II and Image Reader LAS‐1000 Plus software (Minato‐ku, Tokyo, Japan). Primary antibodies included protein kinase B (AKT), ERK1/2, beta‐actin (antibodies at 1:2000 dilutions), and phosphorylated (p)AKT, pERK1/2 (antibodies at 1:1000 dilutions; Cell Signaling Technology). Peroxidase labeled anti‐rabbit or anti‐mouse secondary antibodies were used (Cell Signaling Technology).