Fig. 4. Regulation of endogenous p38α activity by MAP kinase kinases. Activated epitope-tagged MKK3b, MKK3, K6(1–18)–K3 and K6(1–82)–K3 (A) or MKK3b, MKK3bΔ, MKK6, MKK6Δ and MKK3 (B) were expressed in COS7 cells. The activated MKKs were constructed by replacing the two sites of activating phosphorylation with glutamic acid residues. The epitope-tagged MKK expression and endogenous p38α were examined by immunoblot analysis of cell lysates. The activity of the endogenous p38α was measured in immune complex kinase assays using ATF2 as the substrate. The phosphorylated ATF2 was detected after SDS–PAGE by auto- radiography and was quantitated by PhosphorImager analysis. The p38 activity is presented as relative protein kinase activity.