Homogenized tumor tissue or cells were lysed in 2% SDS containing protease inhibitor cocktail (Sigma, p8340) and phosphatase inhibitor (GenDEPOT, P3200). Equal amounts of protein per sample were subjected to SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked in 5% skim milk in TBST (TBS containing 0.1% Tween 20) for 30 min. Primary antibodies were incubated overnight at 4°C in 3% BSA in TBST. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in 5% milk and TBST for 1 h at room temperature. Bands were visualized using Pierce ECL Plus Western blotting substrate (ThermoFisher 32132). Protein expression was quantified by measuring the pixel intensity of each band using ImageJ. pFAK/FAK represents the ratio of the pFAK signal to the total FAK signal of each sample.
Antibodies used were as follows: rabbit anti-DDR1 (Cell Signaling Technology, 5583), mouse anti-FAK (BD Biosciences, 610088), rabbit anti-pFAKY397 (Cell Signaling Technology, 8556), and mouse anti-β-actin (Sigma, A5441).
Antibodies used were as follows: rabbit anti-DDR1 (Cell Signaling Technology, 5583), mouse anti-FAK (BD Biosciences, 610088), rabbit anti-pFAKY397 (Cell Signaling Technology, 8556), and mouse anti-β-actin (Sigma, A5441).