P egfr

Cultured cells were washed with PBS twice and then lysed in 1× Laemmli sample buffer (Bio-Rad) directly. Lysates were heated at 100 °C for 10 min and centrifuged at 14,000 rpm for 10 min before loading to the 4–15% precast gels (Bio-Rad). Samples were run under 80 V for 15 min, then switched to 130 V for another 1 h, and then transferred to PVDF membranes at 100 V for 90 min. Membranes were blocked in a 5% skim milk PBST buffer for 30 min at room temperature, followed by primary antibody incubation overnight at 4 °C. HRP-conjugated secondary antibody was then applied and incubated for 2 h at room temperature. Membranes were imaged using a ChemiDoc MP Imaging system (Bio-Rad) and organized with Image Lab software. Primary antibodies: EGFR (CST #4267S); p-EGFR (CST #3777S); AKT (CST #9272S); p-AKT (CST #4058S); PI3K (CST #4257S); p-PI3K (CST #4228S); BRAF (CST #9433S); p-BRAF (CST #2696S); MEK (CST #9126S); p-MEK (CST #9121S); ERK (CST #4695S); p-ERK (CST #4370S); CRBN (CST #71810S); VHL (CST #68547S); GAPDH (GeneTex, GTX #110118), IgG (CST #3900S).