In vitro kinase assay

MDA-MB-231 cells were incubated in the absence or presence of 10 μM of Imatinib in serum-free media as indicated. Afterward, the resulting nuclear and cytoplasmic fractions were isolated, at which point 15 μg of each fraction was incubated in kinase assay buffer¹⁰ supplemented with 2 μg of the artificial c-Abl substrate, GST-Crk (Product # 14-468, EMD Millipore, Billerica, MA, USA). The protein kinase reactions were initiated by the addition of ATP (5 μM) and allowed to proceed under continuous rotation for 60 min at 37 °C. The reactions were terminated by addition of 4 × sample buffer⁴² and subsequently prepared for anti-phospho-Y221-Crk (1:500; Abcam, Cambridge, MA, USA) immunoblotting to monitor c-Abl kinase activity. Total anti-Crk (1:1000; Abcam, Cambridge, MA, USA) antibody was used as a loading control.