(A) Phosphorylation of WASP by Btk in a reconstitution system. Cotransfection of pcDNA3/WASP with pEF-BOS vector alone (lane 1), pEF-BOS/wild-type (WT) Btk (lane 2), pEF-BOS/Btk (K430R) (lane 3) or pEF-BOS/Btk (WW251LL) (lane 4) into 293T cells was performed with lipofectamine. Cells were harvested after 48 hours and lysed with Triton X-100 lysis buffer. After preclearance, lysates were immunoprecipitated with the anti-WASP antibody 503 and immunoblotted with the antiphosphotyrosine (APT) antibody 4G10 (top). The same membrane was reprobed with the anti-WASP antibody 503 (middle). To detect the expression of Btk constructs, the total cell lysates (TCL) were also loaded and immunoblotted with the anti-Btk antibody 43-3B (bottom). (B) In vitro kinase assays of wild and mutant Btk. The expression vectors of Btk (WT) (lane 1), Btk (WW251LL) (lane 2), or Btk (K430R) (lane 3) were transfected into 293T cells. Cells were harvested after 48 hours, and the lysates were immunoprecipitated with the anti-Btk antibody 48-2H. The in vitro kinase assay was performed with denatured enolase as a substrate. The bottom panel demonstrates the expression of Btk protein in the lysates by immunoblotting with the anti-Btk antibody 43-3B. (C) Association of WASP with wild-type or mutant Btk. Transfection of pEF-BOS/wild type (WT) Btk (lane 1) or cotransfection of T7 epitope-tagged WASP with pEF-BOS/wild-type (WT) Btk (lane 2), pEF-BOS/Btk (K430R) (lane 3), or pEF-BOS/Btk (WW251LL) (lane 4) into 293T cells was performed with lipofectamine. Cells were harvested after 48 hours and lysed with digitonin lysis buffer. After preclearance, lysates were immunoprecipitated with the anti-T7 tag antibody and immunoblotted with the anti-Btk antibody 43-3B (top) or with the antiphosphotyrosine (APT) antibody 4G10 (second), followed by reprobing with the anti-WASP antibody 503 (third). To detect the expression of Btk proteins, the total cell lysates (TCL) were also loaded and immunoblotted with the anti-Btk antibody 43-3B (bottom).