Figure 5

Phosphorylation of EF-1γ bound to the 26S proteasome (A) Phosphorylation of p48 complex and the 26S proteasome by MPF. Purified p48 complex and the 26S proteasome from immature oocytes were treated with or without MPF as indicated in the presence of [γ-³²P]-ATP. After incubation at 30°C for 1 hour the reaction was stopped by addition of SDS-PAGE sample buffer. ³²P-labeled proteins were resolved by SDS-PAGE followed by autoradiography on Imaging plates (Fuji Film). (B) Immunoprecipitation of p48 complex by anti-EF-1 complex. The 26S proteasome fractions were treated with affinity purified anti-EF-1 complex (Anti) or control IgG (Cont) and EF-1 components were immunoprecipitated as described previously [37]. Supernatants (S) and precipitates (P) were resolved by SDS-PAGE followed by autoradiography. Arrows indicate ³²P-labeled 48, 37 and 30 kDa protein bands. Image analysis was performed using a Molecular Imager FX (BioRad).