E2F3a occupies the Cdc6 promoter in myoblasts, but it is replaced by E2F4 after differentiation. (A) ChIP of C2C12 myoblasts cultured in GM with antibodies (Ab) detecting E2F1, E2F3a, and E2F4. As a control, ChIP was also performed in parallel with normal rabbit IgG (NR IgG). Purified DNA from enriched chromatin fragments was amplified by PCR with the E1 primers. (B and C) ChIP of C2C12 myoblasts cultured in DM at the indicated times using normal rabbit IgG and either anti-E2F3a or anti-E2F4, respectively. Precipitated DNA was analyzed for the presence of the Cdc6 promoter by PCR. (D) The occupation of the Cdc6 promoter by E2F4 correlates with its silencing in differentiated myoblasts. Immunofluorescent staining of C2C12 myoblasts cultured in GM or DM for 96 h was performed by using a Cdc6-specific antibody. Antibody staining is visualized as red. RT-PCR detection of Cdc6 transcripts in C2C12 myoblasts cultured in GM or in DM for 72 or 96 h. GAPDH expression is used as a loading control. White lines indicate that intervening lanes have been spliced out. Bars, 20 µm.