Immunoprecipitation and Western Blot Analysis of Signaling Proteins

Cells were lysed in PSB buffer and protein concentration was determined as described previously [50 (link), 51 (link)]. For immunoprecipitation, cell lysates were incubated with antibodies at 4°C overnight. The immune complexes were incubated with protein G/A bead flurry for another two hours at 4°C. Samples were then heated and separated on 8% SDS-PAGE gels, and membranes were incubated with specific antibodies as listed in supplemental methods. Antibodies used were anti-ABL (8E9; BD Bioscience), anti-phospho-ERK (Cell signaling Technology), anti-GRB2 (Santa Cruz Biotechnology), anti-phospho-Y177 BCR (Cell Signaling), anti-STAT5 (Cell Signalling), anti-phopho-STAT5 (Cell Signaling), anti-phospho-CRKL (Cell Signaling), and anti-phosphotyrosine antibodies (4G10; Millipore).