LAT adapter protein is required for coupling CD2 to ERK1/2 activation.

The LAT-deficient Jurkat cells ANJ3 and ANJ3 cells reconstituted with wild-type LAT (ANJ3-LATwt) (10⁶ cells/test) were either left unstimulated, stimulated with T11₂ + T11_3, OKT3 or PMA (10 ng/mL) plus ionomycin (1.5 μM) (P + I) at 37°C for the indicated periods of time and lysed in 1% Brij97 lysis buffer. Clarified PNLs (0.3 × 10⁶ cell equivalents) of unstimulated and stimulated cells were resolved by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with an antiphospho-ERK1/2 antibody specific for the dual phosphorylated (Thr202 and Tyr204) activated p44 and p42 MAPK (Erk1 and Erk2) (upper panel). The same membrane was stripped and reblotted with anti-ERK1/2 antibody (lower panel).