Imagequant las 4000 instrument

Cells were incubated overnight without savolitinib, treated with 2 μM savolitinib for four hours and then lysed and subjected to the PathScan^® RTK Signaling Antibody Array exactly as instructed by the manufacturer (catalog# 7982S, CST, Inc, Bedford, MA). Chemiluminescence was captured on an ImageQuant LAS 4000 instrument (Fuji). Densitometry analysis was performed using the ImageQuant TL Array software, V8.1 (GE Healthcare).

Cell proteins were extracted by lysis buffer (Cell Signaling, USA) with a complete protease inhibitor cocktail (Sigma, USA) on ice. Protein concentrations were quantified using a BCA protein assay kit (Beyotime, China). Eighty micrograms of protein sample was mixed with 5 × loading buffer and loaded into the lanes of 10% sodium dodecyl sulfate–polyacrylamide gel (SDS). The proteins were separated by electrophoresis and transferred to 0.2 μM PVDF membranes (Millipore, USA). After blocking with 5% nonfat milk (Sigma, USA) for 2 h and washing with TBS-T three times at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight. After washing with TBS-T for three times, the membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were detected using an ECL detection reagent (Millipore, USA) and imaged by the Image Quant LAS-4000 instrument (Fujifilm, Japan). The band density was analysed by ImageJ and normalized to the level of β-actin in each group. The experiments were repeated independently for three times.