Figure 4

Several signaling pathways are likely to control Myf5 mitotic degradation (A) 1 μl of untreated (upper panel) or dephosphorylated (deP, lower panel) Myf5 was incubated with buffer (lane 2), recombinant CDK1/cyclin B (New England Biolabs, 40U, lane 3), recombinant Erk2 (New England Biolabs, 200U, lane 4) or both kinases (lane 5) for 30 min at 30°C. The reaction mix was resolved by 10% SDS-PAGE. "Start" is a non-incubated control. (B) Myf5 degradation is sensitive to very low concentrations of calcium: a mitotic extract was prepared using XB without CaCl₂ but supplemented with 6 mM EGTA (see Methods), then incubated with 0.1 mM CaCl₂ and 6 mM EGTA, or an equivalent volume of water as a control, for 10 min at 25°C. 2 μl of Myf5 were incubated for 1 hour with 18 μl of treated extract. 2 μl samples were resolved by 10% SDS-PAGE at the beginning and the end of the reaction. Myf5 degradation rate was quantified as previously described; the graph represents mean values of 3 experiments. Bars represent standard deviations.