Untreated (–) and treated (+) A431 whole cell extracts (30 μg) were separated by 5% SDS-PAGE, and the membrane was blotted with EGFR (phospho Thr669) antibody [HL3264] (GTX640911) diluted at 1:1000 and EFGR antibody (GTX640663) diluted at 1:1000. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody.

Specificity test was performed by coating the plate with EGFR peptide and EGFR (phospho Thr669) peptide (15.63 ng/mL). Coated peptides were probed with EGFR (phospho Thr669) antibody [HL3264] (GTX640911) (1 μg/mL). Goat anti-rabbit IgG antibody (HRP) (GTX213110-01) (1:10000) was used to detect the bound primary antibody.