c-Abl is required for efficient HS1 phosphorylation in response to TCR engagement. (A) Jurkat T cells were pretreated with or without 10 μM imatinib mesylate for 1 hour, and then stimulated with C305 for indicated times. Lysates were immunoprecipitated with anti-CrkL or anti-HS1, and immunoblotted with indicated antibodies. Phosphorylation of ZAP-70 at tyrosine 319 was assessed by immunoblotting of whole-cell lysates. (B) Jurkat T cells were transfected with control vector or shAbl-3 and cultured for 48 hours to suppress c-Abl expression. Cells were stimulated with or without SEE-pulsed Raji B cells for 1 minute, and lysates were immunoprecipitated with anti-HS1 and immunoblotted with the indicated antibodies. Abl expression and ZAP-70 phosphorylation was assessed by immunoblotting of whole-cell lysates.