TCR stimulation with or without costimulation activates p38α through the alternative pathway. (A) T cells purified from spleen and lymph nodes of wild-type (WT) or p38α^Y323F mice were stimulated with plate-bound anti (α)-CD3 and/or α-CD28 antibodies (Abs) for 30 minutes. Whole lysates from 4 × 10⁶ cells were immunoblotted with antibodies recognizing p38 phosphorylated on Tyr323 (P-Y323-p38), Thr180/Tyr182 (P-p38), or total p38. (B) WT and p38α^Y323F T cells were stimulated with α-CD3/CD28 Abs, PMA, or sorbitol, and phospho-p38 was detected in whole cell lysates. (C) p38α or (D) p38β were immunoprecipitated from WT and p38α^Y323F T cells stimulated with α-CD3/CD28 Abs or PMA, and in vitro kinase (IVK) assays of immune complexes with ATF-2 as a substrate were performed. (E) B cells purified from spleen of WT or p38α^Y323F mice were stimulated with anti-IgM antibody (10 μg/mL) for 10 minutes, p38α specifically immunoprecipitated from whole cell lysates, and kinase assays performed as in panel C.