p38 MAPK is activated by ligation of the TCR on thymocytes and Thy278/107 cells.
A, Lysates were prepared from unstimulated (U) or anti-CD3-stimulated thymocytes at the indicated times after stimulation. Active p38 MAPK in the lysates was detected by immunoblotting with a phospho-specific antibody to the dually phosphorylated form of p38 MAPK (upper). The membrane was stripped and reprobed with a polyclonal antibody to p38α MAPK to test for equal protein abundance (lower). B, Thy278/107 cells were stimulated with anti-CD3 MAb for increasing times, after which cells were lysed. Phospho-p38 and total p38α MAPK in the lysates were detected as in A (upper and middle). In vitro kinase assay reactions were performed as described in Materials and Methods using GST-ATF-2 as substrate. Phospho-ATF-2 was detected by immunoblotting with an antibody against Thr71-phosphorylated ATF-2 (lower).
