In vitro inhibition of p-EGFR and relative pathways

HCC827 cells were seeded on cell slides for 24 h and treated with BCGN or gefitinib (2 μg/mL) for 6 h. Then cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 10% serum for 45 min at room temperature. Next, cells were labeled with Phospho-EGFR (Tyr1068) Rabbit Polyclonal Antibody at room temperature for 1.5 h and labeled with Alexa Fluor® 488 AffiniPure goat anti-rabbit IgG (H + L) for 1 h at room temperature. F-actin was stained with phalloidin-tetramethylrhodamine (TRITC) (Ex/Em: 540 nm/560 nm) and nuclei were stained with DAPI.

For Western blot assay, HCC827 cells were seeded on cell slides for 24 h and treated with BCGN, gefitinib (2 μg/mL) or PBS for 6 h. Then cells were extracted by adding RIPA Lysis solution and phenylmethanesulfonyl fluoride (PMSF). Then proteins were quantified by BCA quantification kit and denatured. Samples containing same concentration of total proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresed protein samples were transferred to polyvinylidene difluoride membranes (Bio-Rad). After washing three times, the membranes were blocked by non-fat dry milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies to mark p-EGFR (Tyr1068), t-EGFR, ERK1/2, p-ERK1/2 (Thr202/Tyr204), AKT1/2/3, p-AKT1/2/3 (Ser473) (1:1,000 dilution). After washing three times, the membranes were incubated for 1 h at room temperature with HRP-conjugated species-specific secondary antibody and then pictures were captured by electrochemiluminescence (ECL) system (Bio-Rad).