Immunoblotting Analysis of Src and FAK Signaling

Cell lysates and electrophoresis was performed as previously described [7 (link)]. Immunoblotting of Src (Y⁴¹⁹; Millipore, USA) [14 (link)], Src kinase (Cell Signaling Technology, USA) [15 ], FAK (Y⁸⁶¹; Invitrogen, UK) [16 (link)] and FAK (Y³⁹⁷; Abcam, UK) [17 (link)] were performed at 1:1000 dilutions with rabbit anti-human antibodies. Proteins were visualized by addition of a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated polyclonal anti-rabbit IgG. HRP-conjugated antibodies raised against beta tubulin were used as loading controls. HRP-conjugated protein was visualized using an enzyme-linked chemiluminescence reaction (Thermo Fisher Scientific, UK) and quantified using a LAS3000 image analyser (Fujifilm, Tokyo) and analysed with Image J software (NIH, US).