Lad associates with Lck and Zap-70 in response to chemokine treatment. (A) Lck associates with Lad upon chemokine treatment. Jurkat T cells that stably express Lad-FLAG (Lad) or were transfected with empty vector (Vec) were activated with anti-CD3 antibody (CD3), SDF-1α (S), or RANTES (R) for 10 minutes. The cell lysates were then subjected to immunoprecipitation with 5 μg antiFLAG antibody, and the precipitates were analyzed by immunoblotting with antiLck antibody (upper panel). The membrane was reprobed with the anti-FLAG Ab (bottom panel). (B) Zap-70 associates with Lad upon chemokine treatment. Jurkat T cells were transfected with pcDNA3.1 or the expression plasmid encoding Lad-FLAG. These cells were then stimulated for the indicated periods with SDF-1α (S) or RANTES (R), after which the cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody and subsequent immunoblotting with anti–Zap-70 antibody (top panel). The membrane was reprobed with the anti-FLAG Ab (bottom panel). (C) The chemokine-dependent phosphorylation of Zap-70 is repressed by expressing hLad siRNA. Jurkat T cells were transfected with empty pSUPER or pSUPER plasmids expressing SiA or SiB and stimulated with SDF-1α (S) or RANTES (R). The cell lysates were then analyzed by immunoprecipitation with anti–Zap-70 antibody and subsequent immunoblotting with anti-phosphotyrosine antibody (4G10) (top panel). The blot was also probed with the anti–Zap-70 antibody (bottom panel). (D) Jurkat T-cell clones that stably express CCR5 were transfected with pSUPER plasmids expressing control siA or siA and stimulated with SDF-1α or RANTES. The cell lysates were analyzed by Western with anti–phosphoZap-70 (Tyr493) or anti–Zap-70 Abs.