Activation of p38-γ signaling represses MyoD activation of Myogenin transcription. (A) Schematic representation of myoblast differentiation, with MyoD binding to the myogenin promoter within 6–12 h of growth factor removal, and maximal MyoD transcriptional activity achieved between 12 and 48 h. (B–J) Immunofluorescent staining for the Myc (C and I) or Flag epitopes (F), and Myogenin (D, G, and J) in C2C12 myoblasts expressing empty vector (B–D), MKK6EE and p38-α (E–G), or MKK6EE and p38-γ (H–J) after 24 h of differentiation. Note the Myogenin-positive nuclei in cells containing activated p38-α (arrows in F and G). Conversely, note the lack of Myogenin staining in cells containing activated p38-γ (arrowheads in I and J). Nuclei were counterstained with DAPI (B, E, and H). (K) Luciferase assays on 10T1/2 fibroblast extracts expressing MKK6EE and p38-γ, or empty vector. MyoDwt or MyoDmut were expressed as indicated. A diagram of the myogenin-luciferase reporter, shown above the graph, contains two canonical E-boxes (E1 and E2), as well as the PME site, which contains the binding site for Pbx-Meis along with a noncanonical E box. Arrows indicate the location of primers for ChIP assays. Error bars represent ±SEM for n = 9. Asterisk denotes significance (P < 0.00002). (L) Quantification of a Myogenin immunoblot, normalized to MyoD protein levels, from 10T1/2 fibroblast extracts expressing MKK6EE, p38-γ, or empty vector. MyoDwt and MyoDmut were expressed as indicated. Note that the original immunoblots are shown in Fig. S2 G. MyoDmut, MyoD [S199A/S200A] mutant; MyoDwt, wild-type MyoD. Bar, 25 µm.