Protein Quantification and Western Blot Analysis

The protein extraction and Western blot analysis were constructed as previously described (15 (link)). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The primary antibodies of Akt (dilution 1:1,000, rabbit polyclonal, no.10176-2-AP), phosphorylated Akt (Ser473) [p-Akt (Ser473), dilution 1:3,000, mouse monoclonal, no.4051S] and ERK1/2 (dilution 1:1,000, rabbit polyclonal, no. 16443-1-AP) were purchased from Proteintech (Chicago, IL, USA). Phosphorylated ERK1/2 (Thr202/Tyr204) [p-ERK1/2 (Thr202/Tyr204), dilution 1:1,000, rabbit, no. 20G11] and CDK6 (dilution 1:2,000, mouse monoclonal, no. 3136) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). GAPDH antibody (dilution 1:2,000, rabbit, no.10493-1-AP) and the secondary antibodies horseradish-peroxidase (HRP)-conjugated goat anti-rabbit IgG (dilution 1:10,000; catalog no. AS014), and HRP-conjugated goat anti-mouse IgG (dilution 1:10,000; catalog no. AS003) were purchased from Abcam (Cambridge, MA, USA). The specifically bound antibodies were detected with enhanced chemiluminescence (ECL) (Millipore Co. Billerica, MA, USA). Images were analyzed using the BIO Photometer (Eppendorf AG, Hamburg, Germany).