Onset of proliferation is delayed in p38α^Y323F T cells stimulated through TCR/CD28. (A) Wild-type (■; WT) and p38 α^Y323F (□; Y323F) T cells were stimulated with plate-bound anti-CD3/CD28 antibodies or with PMA plus ionomycin (PMA/Iono) for the indicated times, fixed, and RNA were stained with pyronin Y. Error bars represent SEM (3 replicates per condition). (B) WT and p38α^Y323F T cells were stimulated with plate-bound anti-CD3/CD28 antibodies for the indicated times, fixed, and DNA (Hoechst 33342) and RNA (Pyronin Y) were stained. The percentages of positive cells are indicated for each upper quadrant (results are representative of 2 independent experiments). (C) WT and p38α^Y323F (Y323F) T cells were purified from spleen and lymph nodes, labeled with CFSE, and stimulated with plate-bound α-CD3/CD28 antibodies or PMA/Iono for indicated times. One of 4 independent experiments is shown. (D) Summary of percentage of nondivided cells and mitotic indexes of WT and p38α^Y323F (Y323F) T cells from panel C, n = 4; bars indicate mean (± SEM); P value was calculated using the Student t test.