Fig. 3. Msx1 upregulates cyclin D1 expression and Cdk4 activity. (A) C2C12 cells were infected with a control retrovirus (Vector) or retroviruses expressing Msx1 or cyclin D1 and grown under normal culture conditions (a, undifferentiated) or conditions that promote differentiation (b-d, differentiated). Scale bar: 0.1 mm. (B,C) Western blot analysis was performed using extracts prepared from C2C12 cells infected with a control retrovirus (Vector) or with retroviruses expressing Msx1 or Msx1A, followed by cell growth under normal culture conditions (undifferentiated) or conditions that promote differentiation (differentiated). The panels show whole-cell extracts probed with the indicated antibodies, except for panels labeled -Cdk4 nuclear or -Cdk2 nuclear, in which nuclear extracts were used. RNA polymerase II (-RNA PolII) is a control for protein loading. (D) Cdk4 and Cdk2 kinase assays were performed using cell lysates from undifferentiated C2C12 cells or 293T cells infected with a control retrovirus (Vector) or those expressing Msx1 or Msx1A. The indicated kinases were immunoprecipitated using the corresponding antisera and kinase assays were performed using GST-Rb (amino acids 379-792) as substrate. Similar results were obtained using lysates prepared from differentiated C2C12 cells (data not shown).