Ecl western blotting detection system

To assess EGFR expression, total protein samples were extracted from HCC827 and NCI-H520 cells in lysis buffer (RIPA) containing protease inhibitor (PMSF). The resulting lysates were centrifuged at 13.2×10³ rpm for 15 min and the supernatants were collected. The protein concentrations were measured using the BCA protein assay kit (Solebo biotech Ltd). SDS–PAGE and Western blotting were performed using 80 μg of proteins. The lysates were resolved by electrophoresis (70 V for 25 min and 110 V for 1.5 h) and transferred onto NC membranes. After blocking in 5% non-fat milk for 2 h, the blots were incubated with the first antibody: rabbit anti-human EGFR monoclonal (1:1000, Abcam) and mouse anti-human GAPDH (1:1500, Abcam) were used as a loading control overnight at 4 °C. The blots were washed and incubated with the second antibody: goat anti-rabbit IgG HRP-linked antibody (1:5000, Cell Signaling Technology); goat anti-mouse antibody (1:5000, Cell Signaling Technology) at room temperature for 1.5 h. Western blot bands were captured by the ECL Western blotting detection system (BD). GAPDH was used as a loading control. After development, the films were scanned with BIO-RAD Gel Doc XRS⁺. The images were opened and analyzed by ImageLab (BIO-RAD) software. Three samples of each tumor cell type were prepared for Western blot to obtain semi-quantitative data for statistical analyses.