ERK signaling during NKT-cell activation. (A) Flow cytometric analysis of CD3 cell surface expression by NKT cells after coincubation with APCs for the indicated times. The results are from 1 representative experiment of 3. Similar results were observed using 2 different clones. (B) NKT cells were exposed to APCs for the indicated times, and then lysed and phospho-ERK was detected by Western blotting (top panels). The same membrane was then stripped and probed with an antibody for total ERK (bottom panels). APCs were pretreated with the U0126 inhibitor before incubation with the NKT cells to eliminate APC phospho-ERK signal. The plot shows densitometric analysis of the phospho-ERK signal from the upper band normalized by the corresponding total ERK signal. Similar results were obtained using 3 different NKT cell clones. (C) Flow cytometric analysis of CD69 expression by NKT cell clones after coincubation with APCs for the indicated times. The plots on the right show the change in mean fluorescence intensity compared with coincubation with untransfected APCs. Similar results were obtained from 2 additional clones.