Immunoblotting

Cells were seeded at a specific cell density in 6-well dishes and cultured overnight in complete medium. Afterwards cells were first pretreated with inhibitors for 20 min to 1 h in serum-free medium and then stimulated with PDGF-BB (20 ng/mL) for the indicated time points and subsequently washed and then lysed in RIPA buffer, supplemented with complete protease inhibitor tablets with EDTA from Roche (Basel, Switzerland) and 0.2 mM vanadate. Protein concentrations in supernatants were determined with the BCA kit (Thermo Fisher Scientific) and equal amounts of proteins were loaded onto Any kD Mini-PROTEAN TGX Precast Gels (Bio-Rad). After SDS-PAGE electrophoresis and transfer, membranes were first blocked in 5% BSA in TBS-tween and then probed with primary antibodies against pSer³-cofilin (1:4000), pan-cofilin (1:10000), tubulin (1:5000), PLCγ (1:1000), p85 (1:1000), and pY857 (1:1000) for 1.5 h at room temperature. Then, membranes were probed with HRP-conjugated donkey anti-rabbit antiserum as the secondary antibody (1:25000) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) detection on a charge-coupled device (CCD) camera (Fuji, Minami-Ahigata, Japan). The protein bands were quantified using Aida Analyzer software. The quantified values of p-cofilin protein bands were subtracted by the quantified values of total-cofilin protein bands, and then they were normalized to the value of the control condition. The quantified data are presented as a bar-chart or written values underneath each corresponding band.