Fig. 5. Regions of Msx1 required for upregulation of cyclin D1 include its transcriptional domains. (A) Schematic diagrams showing the primary structures of Msx1 and Msx2; the percent identity in the N-terminal region, homeodomain and C-terminal regions are indicated. Also shown are the protein regions contained in the truncated Msx1 derivatives, Msx11, Msx12 and Msx13. (B) Western blot analyses were performed using extracts prepared from differentiated C2C12 cells following infection with a control retrovirus (Vector) or with retroviruses expressing the indicated Msx protein. Western blots were probed with -cyclin D1 or -MHC antibodies; -RNA PolII is a control for protein loading. (C) C2C12 cells were infected with a control retrovirus (Vector) or a retrovirus expressing an Msx1-estrogen receptor fusion protein (Msx11-ER). Infected cells were incubated in the presence (+) or absence (-) of tamoxifen (100 nM) and grown under conditions that promote differentiation. Scale bar: 0.1 mm. (D) Ribonuclease protection analyses were performed using total RNA prepared from C2C12 cells that had been infected with a control retrovirus (Vector) or with a retrovirus expressing Msx11-ER. Infected cells were treated with tamoxifen (100 nM) for the indicated times and ribonuclease protection assays were performed using a cyclin D1 riboprobe or an RPL32 riboprobe as a control for RNA loading.