Western blotting and antibodies

SDS-PAGE gels were transferred onto nitrocellulose 0.2 μm membranes (Amersham^TM). Transferred membranes were immersed in blocking solution (10 mM Tris pH 8, 150 mM NaCl, 5% weight/volume (w/v) skimmed powder milk) for 60 min. After blocking, membranes were washed three times with TBS-T (10 mM Tris pH 8, 150 mM NaCl, Tween-20 0.1% v/v) prior to incubation with primary antibody solution (TBS-T with BSA 5% w/v) o/n at 4 °C shaking. Antibodies used were: phospho-Src Tyr 216 (CSB-{"type":"entrez-nucleotide","attrs":{"text":"PA050132","term_id":"2069200069","term_text":"PA050132"}}PA050132), phospho-Src Tyr 419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44–662G and CST#2105) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000–1:5000. After incubation with the first antibody, membranes were washed with 20 ml TBS-T three times and immersed in secondary antibody solution (TBS-T skimmed powder milk 5% m/v). Secondary antibodies anti-rabbit or -mouse IgG DyLight conjugate at 680 or 800 nm (CST #5366, #5151, #5470, #5257) were used at double the dilution factor of the primary during 1 h at room temperature protected from light. After incubation with secondary antibodies, membranes were washed several times with TBS-T. Membranes were scanned in an Odyssey CLx scanner.