CXCL12 induces tyrosine phosphorylation of ZAP-70 and Vav1.

(A) Jurkat cells were preincubated for 3 hours in the presence of the carrier alone or of the ZAP-70 inhibitor piceatannol (25 μM) and were stimulated with CXCL12 (50 ng/mL) for 1 minute at 37°C. Antiphosphotyrosine immunoblot analysis was then performed on total cell lysates. (B) Immunoprecipitations with anti–ZAP-70 antibody followed by antiphosphotyrosine immunoblot analysis was performed on ZAP-70⁺-P116 and P116 cells activated by CXCL12 (50 ng/mL) for the indicated periods of time. The membrane was then stripped and reblotted with an anti–ZAP-70 antibody. (C) Immunoprecipitations with anti-Vav1 and anti-Lck antibodies, followed by antiphosphotyrosine immunoblot, were performed on Jurkat, ZAP-70⁺-P116, and P116 cells activated by CXCL12 (50 ng/mL) for 1 minute. Membranes were stripped and reblotted with anti-Vav1 and anti-Lck antibodies.