p56^lck is required for PECAM-1 tyrosine phosphorylation in T cells.

Wild-type Jurkat (top), p56^lck-deficient JCaM1.6 (middle), or p56^lck-reconstituted JCaM1.6 (bottom) T cells were treated with no Ab (None; lane 1), 1 μg/mL of murine antihuman CD3ε (CD3; lane 2), 50 μg/mL of murine Ab specific for human PECAM-1 (PECAM, lane 3), or both anti-CD3ε and PECAM-1.3 (CD3 + PECAM; lane 4) at 4°C. After prewarming for 10 minutes at 37°C, receptor cross-linking was induced by addition of 100 μg/mL of GAM F(ab′)₂. After a further incubation for 3 minutes, cells were lysed in Triton X-100, and PECAM-1 immunoprecipitates (IP PECAM-1) were prepared. Immunoblot analysis was performed to determine the extent of PECAM-1 tyrosine phosphorylation, using an antiphosphotyrosine Ab (PY20; top panels) and the amount of PECAM-1 antigen present in the immunoprecipitates using the PECAM-1.3 Ab (αPECAM-1; bottom panels). Note that PECAM-1 does not become tyrosine phosphorylated in the absence of p56^lck and that reconstitution of p56^lck expression restores PECAM-1 tyrosine phosphorylation.