(A) Association of Btk and BLNK in 293T cells. Flag-BLNK and Btk [wild-type or SH3-mutated (WW251LL)] were coexpressed with (+) or without (−) Syk. Coprecipitation of Btk with Flag-BLNK was detected by immunoprecipitation (IP) with the anti-Flag antibody followed by immunoblotting with the anti-Btk antibody 43-3B (top panel). (Without the expression of Flag-BLNK, Btk protein was not detected with the same procedure; data not shown.) The equality of the amounts of the Flag-BLNK protein in each immunoprecipitate was confirmed by reprobing the same filter with the anti-Flag antibody (second). Tyrosinephosphorylation of Flag-BLNK by Syk in precipitates was confirmed by immunoblotting with the antiphosphotyrosine (anti-pTyr) antibody 4G10 (third), and the equality of the amounts of the Btk protein in cell lysates was confirmed by immunoblotting with the anti-Btk antibody 43-3B (bottom). (B) Association of Btk and BLNK in DT40 cells. After DT40 cells, in which T7-Btk was stably expressed, were stimulated with the anti-chicken IgM antibody M4 for the indicated time periods, the cells were lysed and immunoprecipitated with the anti-T7 antibody. The coprecipitation of the endogenous BLNK was detected by immunoblotting with the anti-BLNK antibody (top panel). The equality of the amounts of T7-Btk protein in each of the immunoprecipitates was confirmed by reprobing the same filter with the anti-Btk antibody 43-3B (second). The tyrosinephosphorylation of BLNK (third) or that of T7-Btk (bottom) was detected by immunoprecipitating with the anti-BLNK antibody or the anti-T7 antibody followed by immunoblotting with the antiphosphotyrosine antibody 4G10.