Figure 3
From: Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5
Hyperphosphorylated Myf5 is stable. An equal volume of reticulocyte lysate containing in vitro translated Myf5 and of Xenopus mitotic egg extract (CSF) were incubated together for 60 min in the presence of 1 μM microcystin LR. At each time point, 0.5 μl samples were analyzed by 10% SDS-PAGE (panel A). After this initial incubation, 2 μl of the reaction mixture containing hyperphosphorylated Myf5 (Myf5PPP) were further incubated in 18 μl of fresh mitotic extract, either in the presence of 1 μM microcystin (panel B) or without inhibitor addition (panel C), or without microcystin and in the presence of the proteasome inhibitor MG132 (200 μM, panel D). At each time point, 2 μl samples were analyzed by 10% SDS-PAGE.