Figure 3.

Reconstitution of p27 ubiquitination activity. (A) SCF^Skp2 and Cul1/Skp1 complexes were prepared from Sf9 cells coexpressing His₆-tagged Cul1, His₆-tagged Skp1, and Skp2 (lane 1), or His₆-tagged Cul1 and His₆-tagged Skp1. Proteins were purified with Ni-NTA resin and analyzed by SDS/PAGE and immunostaining with antibodies against Cul1, Skp1, and Skp2. (B) Ubiquitination reactions containing [³²P]-GST-p27, 30 μM ubiquitin, 100 nM E1 (ubiquitin activating enzyme), 2 μM ubiquitin aldehyde, 4 μM MG273, 1 μM okadaic acid, and 2 mM ATP were carried out by using in the absence (lanes 1 and 2) or presence of SCF^Skp2 (lanes 3–6) or Cul1/Skp1 (lane 7). Reaction mixtures were further supplemented with Nedd8 pathway components (0.29 μM Nedd8, 1 μM Nce1, and 100 nM Nae1; lanes 2, 4, 6, and 7) and 1 μM Cdc34 (lanes 2 and 5–7).