Lck kinase expression is required for LAT tyrosine phosphorylation on CD2 stimulation.

(A) The Lck-deficient JCaM 1.6 cells reconstituted with wild-type Lck were either left untreated or treated with tetracycline (Tet) 1 μg/mL for 8 days. Lysates from equivalent cell numbers (0.3 × 10⁶) were immunoblotted for Lck to confirm the tetracycline-regulated Lck expression. (B) Prior to stimulation, the surface expression of both CD3 and CD2 was evaluated in either untreated or Tet-treated cells by direct immunofluorescence. Cells were then either left unstimulated or stimulated via either CD2 or CD3 for the indicated periods of time. Cell lysates were subjected to LAT immunoprecipitation. LAT tyrosine phosphorylation is shown (C, upper panel). The same membrane was stripped and reblotted with anti-LAT antibody (C, lower panel).