Lad is required for the chemokine-induced activation of focal adhesion molecules such as Pyk2 and paxillin. (A) Jurkat T-cell clones expressing CCR5 were transfected with pSUPER plasmids expressing control siA or siA and treated with SDF-1α (S) or RANTES (R) for 2 or 5 minutes. Pyk2 was immunoprecipitated and the immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine or anti-Pyk2 antibody. IgG was used as a negative control. (B) Jurkat T cells stably expressing Lad or the SH2 domain of Lad were treated with SDF-1α or RANTES and processed as described in panel A. (C) The cell lysates prepared as described in panel A were analyzed by immunoprecipitation with anti-paxillin antibody and subsequent immunoblotting with anti-phosphotyrosine antibody (4G10) (upper panel). As a protein-loading control, the blots were reprobed with anti-paxillin antibody (lower panel). (D) The same cell lysates used in panel A were analyzed for the Lad levels by Western blotting with anti-Lad antibody (upper panel) and with anti-actin antibody (lower panel).