A, upper-left panel: Jurkat cells were stimulated or not with the anti-CD3ε mAb UCHT1 for 10 minutes, lysed, precleared and GST or GST-SAP pull down experiments were performed. Upper-Middle panel: Jurkat cells were treated with 25 µM of pervanadate for 30 minutes, lysed, precleared and GST-SAP pull-down was performed as above. Upper-Right panel: T-cell blasts were stimulated with UCHT1 for 10 minutes, lysed, precleared and GST-SAP was incubated with cell lysate. Pull downs were immunoblotted with anti-phosphotyrosine (4G10) antibody. B, Jurkat cells were either untreated or treated with 25 µM of pervanadate for 30 minutes, lysed, precleared and GST or GST-SAP were incubated with cell lysates. GST pull downs were immunoblotted for NTB-A or CD3ζ, as indicated. C, T-cell blasts were stimulated or not with UCHT1 for 10 minutes, lysed, precleared and GST or GST-SAP pull downs were performed and immunoblotted for CD3ζ. These experiments were repeated at least 3 times. The position of NTB-A is indicated by an arrow.