Protein Expression Analysis Protocol

Extraction and concentration determination of sample protein were performed as previously described^{41 (link)}. Equal amount of protein from different samples was loaded on SDS polyacrylamide gels, then were transferred to nitrocellulose membrane. After blocking with Tris-buffered saline (TBS) containing 5% fat-free milk, the blots were incubated with primary antibodies overnight at 4 ℃ and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz, CA, USA) for 1 h at room temperature. Rabbit antibodies against p-AKT (473), p-AKT (308), AKT, and p70S6K, and mouse antibody against p-p70S6K were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against EGFR, HER2, HER3, acetyl-α-tubulin, cyclin D, cyclin E, CDK2, CDK4, CDK6, and actin were purchased from Santa Cruz (USA). Antibodies against p-HER2 (Tyr1248) and p-HER3 (Tyr1289) were purchased from Affinity (China). Antibodies against p-EGFR (Tyr 1069) and α-tubulin were purchased from Abscience (China). Subsequently, the blots were washed with TBST for three times, incubated with ECL reagent (Millipore, USA), and visualized by ChemiDoc XRS+ gel imaging system (Bio-Rad, USA). Image gray value analysis was performed by ImageJ (NIH, USA) and the intensities of actin were used as a control for all other bands^{40 (link)}.