4.2. Western Blot Analysis

The harvested cells were lysed in RIPA buffer (Thermo Scientific, Boston, MA, USA) containing protease inhibitors and phenylmethylsulfonyl fluoride (Thermo Scientific, Boston, MA, USA). The lysates were centrifuged for 20 min at 7500 rpm at 4 °C and the supernatant was collected. The lysates were quantified using the BCA Protein Assay Kit (Thermo Scientific, Boston, MA, USA). Equal amounts of protein (30 μg) were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). The membranes were blocked in 5% skim milk in TBS-T (10 mmol/L Tris-HCl, 50 mmol/L NaCl, and 0.25% Tween-20) for 1 hour at room temperature. The membranes were incubated with anti-phospho-FGFR, anti-phospho-FRS, anti-phospho-ERK1/2, anti-pan ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), anti-FGFR1 (Proteintech, Rosemont, IL, USA), anti-FGFR2, anti-FGFR3, anti-cleaved caspase-3, anti-cleaved PARP (Abcam, Cambridge, UK), anti-FRS and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies overnight at 4 °C. The membranes were washed in TBS-T and incubated with horseradish peroxidase-conjugated (HRP) secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). Antibody-bound proteins were detected using LAS-3000 (Fujifilm, Tokyo, Japan) with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Boston, MA, USA).