Figure 3.

MyoD protein stability is affected by Pax7. (A) Western blots from C3H10T1/2 cell lysates normalized to equivalent levels of protein (lysate aliquots from a representative experiment equivalent to those used in Fig. 2, B and C) were probed for MyoD upon coexpression with Pax7, ΔN, and ΔHD Pax7-deletion mutants (top). Relative levels of Pax7 and Pax7 mutants were monitored (using anti-Pax7 and anti–myc-tag antibodies, respectively) in the same extracts (bottom). (B, left) MyoD protein is recovered to control levels (lane 1) after incubation with MG132 (lane 2) even in the presence of Pax7 (compare lanes 2 and 3). Incubation with DMSO has no effect on MyoD levels in the presence of Pax7 (lane 3, control). (bottom) Quantification of lanes 1–3. (right) MyoD levels are unaffected by Pax7 coexpression under proliferation conditions (compare lanes 4 and 5 to 6 and 7, respectively). (C) MG132 treatment results in partial rescue of MyoD protein (yellow arrowheads) in Pax7-overexpressing adult primary myoblasts (compare to DMSO control; white arrowheads). Bar, 10 μm. (D) MyoD transcriptional activity in C3H10T1/2 cells is not recovered by proteasome inhibition (tested as in Fig. 2 A). Error bars indicate standard deviation. Basal reporter activity was normalized to 1. (E) MG132 treatment results in high levels of MyoD and Pax7 coexpression in the majority of transfected C3H10T1/2 cells (white arrows). In control (DMSO) treated cells, only high levels of Pax7 (arrowheads) or MyoD (asterisks) can be detected in single cells. Bar, 12 μm.