Figure 7
From: Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5
Myf5 ubiquitylation is controlled by phosphorylation. (A) Scheme of Myf5 E3 purification : for details see Methods section. (B) 2 ml of mitotic extract were fractionated as described in A; the fractions obtained from the UnoQ column were screened for their ability to ubiquitylate radiolabeled Myf5 in the presence of a ubiquitylation mix containing GST-Ub (see Methods). The reactions were resolved by 10% SDS-PAGE. (Ub)n-Myf5 indicates poly-ubiquitylated forms of Myf5. (C) The fraction containing the E3 was concentrated about 10-fold using a Centricon 10 K (Millipore). Untreated Myf5 (lanes 1 to 3) or dephosphorylated (deP) Myf5 (lanes 4 to 6) was incubated with the ubiquitylation mix alone (lanes 2, 5) or together with 6 μl of concentrated fraction containing the E3 (lanes 3, 6) for 30 minutes at 25°C. Non incubated Myf5 was loaded in lanes 1 and 4 (START) as control. Similar amounts of radioactive Myf5 and dephosphorylated Myf5 were used.