Other mechanisms of p38α/β activation occur in the absence Cdo/Bnip-2 signaling. (A) Photomicrographs of C2C12 cells stably transfected with control vector or vectors expressing Cdo or Bnip-2 siRNA that were cultured in DM plus SB203580 (+SB) or DMSO vehicle (−SB), fixed, and stained with an antibody to MHC. Bar, 0.1 mm. (B) Quantification of the percentage of nuclei in cultures shown in A that were in MHC⁺ or MHC⁻ cells. Control vector cells are indicated by the negative sign for both Cdo and Bnip-2 siRNA. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from vector control, P < 0.01. (C) Western blot analysis of cultures shown in A. Extracts were probed with antibodies to MHC or total p38α/β (p38). (D) Myoblasts of the indicated Cdo genotype were treated with 0.9 M NaCl or 10 ng/ml TNFα for 15 min and analyzed for pp38α/β and p38α/β. (E) C2C12 cells stably expressing siRNA against Cdo were treated with NaCl or TNFα and analyzed as in D. (F) C2C12 cells stably expressing siRNA against Bnip-2 were treated with NaCl or TNFα and analyzed as in D. (G) Model of Cdo-mediated p38α/β activation during myogenic differentiation. Cdo binds directly to JLP and, via JLP, to p38α/β. Cdo also binds to Bnip-2 and, via Bnip-2, Cdc42. Formation of a Cdo–Bnip-2–Cdc42 complex promotes or stabilizes activation of Cdc42, which in turn triggers signals culminating in phosphorylation and activation of p38α/β bound to Cdo via JLP. Cdo interacts with itself (Kang et al., 2003) and is shown as a dimer. JLP and Bnip-2 are shown as binding to different Cdo proteins of the dimer for convenience and does not preclude the possibility that JLP and Bnip-2 bind the same Cdo protein simultaneously. See text for additional details.