C digit

Cells and tissues were lysed in RIPA solution with protease inhibitor PMSF (1 mmol/L) for 30 minutes on ice. Cell or tissue lysates were centrifuged at 12 000g for 15 minutes at 4°C, and the supernatants were collected. Protein concentrations were quantified using the BCA Protein Assay according to the manufacturer's instructions. Equal amounts (20 μg) of total protein were separated by SDS‐PAGE gel (10%‐12%) at 70 V for 0.5 hour, 120 V for 1 hour and transferred to a 0.45 μm PVDF membrane at 300 mA for 60‐150 minutes. After blocking with 5% non‐fat milk in TBST buffer for 1 hour at room temperature, the membranes were incubated with primary antibody at 4°C overnight. The membranes were washed three times with TBST buffer and then incubated with peroxidase (HRP)‐conjugated secondary antibody for 1 hour at room temperature. Specific antibody binding was detected by the Chemiluminescence Kit (Millipore). Fluorescent signals were detected by a luminescent image analyzer (C‐Digit, Gene Company Limited).

Cells were cultured in 6-well plates at a density of 5 × 10⁵/mL per well and then treated with vemurafenib (10 μM) or/and E3330 (50 μM) for 24 h. The cells were washed with cold PBS three times and lysed in a RIPA solution (Solarbio LIFE SCIENCES) with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, Solarbio LIFE SCIENCES) (1 mM) for 30 min on ice. The supernatants were collected after cell lysates were centrifuged at 12,000 rad for 15 min at 4 °C. The protein concentrations were quantified using the BCA Protein Assay (Solarbio LIFE SCIENCES) according to the manufacturer’s instructions. Equal amounts (30 μg) of total protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10–12%) and transferred to a 0.45-μm PVDF membrane. After blocking with 5% bovine serum albumin (BSA) in TBST buffer for 2 h at room temperature, the membranes were incubated with a primary antibody at 4 °C overnight. The membranes were washed three times with TBST buffer and then incubated with peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Specific antibody binding was detected with the Chemiluminescence Kit (Millipore, Plano, TX, USA). Fluorescent signals were detected by a luminescent image analyzer (C-Digit, Gene Company Limited, China).