Constitutive tyrosine-phosphorylation of WASP in NALM6 cells. (A) Serum-starved 5 × 10⁷ cells (NALM16 and NALM6) were lysed with the NP-40 lysis buffer and, after preclearance, lysates were incubated with GST alone or BtkSH3-GST fusion protein bound to glutathione-Sepharose 4B beads at 4°C for 2 hours. After washing, the beads were boiled with 2× SDS loading buffer and samples were fractionated by SDS-PAGE (5% to 15% gradient gel) and immunoblotted (IB) with the antiphosphotyrosine (APT) antibody 4G10. The asterisk indicates Cbl (120 kD). Molecular mass standards are shown in kilodaltons. (B) The same membrane was reprobed with the anti-WASP antibody 503 (top). Total cell lysates (TCL) of NALM16 and NALM6 cells were loaded and immunoblotted with the anti-WASP antibody 503 (bottom). (C) NALM16 and NALM6 cells (1 × 10⁸) were lysed with the NP-40 lysis buffer and, after preclearance, lysates were immunoprecipitated (IP) with the anti-WASP antibody 503 (lanes 3 and 4) or preimmune rabbit IgG as a control (lanes 1 and 2). Immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with the antiphosphotyrosine (APT) antibody 4G10 (top), followed by reprobing with the anti-WASP antibody 503 (bottom).