MyoD is expressed before Cdc6 after C2C12 myoblasts or primary satellite cells transition from quiescence. (A) C2C12 myoblasts were cultured either in GM or in methionine-free medium (−Met) with 1% serum for 36 h and then restimulated with GM for the indicated times. For Western blot analysis, whole cell extracts were prepared, resolved on SDS-PAGE, and then probed with antibodies specific to MyoD, Cdc6, and GAPDH. MyoD and Cdc6 mRNAs were measured by RT-PCR. GAPDH was used as the internal control. White lines indicate that intervening lanes have been spliced out. (B) Isolated extensor digitorum longus myofibers were cultured in basal medium (0 h) before switching to mitogen-rich medium for the activation of satellite cells. The myofibers were fixed at the indicated times and then coimmunostained for Pax7 (green) and MyoD (red) or Cdc6 (red). Immunostaining for Pax7 identifies the satellite cells (Buckingham, 2007). Arrows point to the individual satellite cells. Approximately 200 myofibers were analyzed in this experiment. Bar, 20 µm.