Figure 4.

UBCH7/UBE2L3 catalyzes ubiquitinylation of p27^Kip1. In vitro ubiquitinylation assay using HELC G₀ lysate and His6-p27^Kip1. Ubiquitinylation was carried out in presence of 500 nM Cdc34/Ubc3/UBE2R1 (Ubc3) or UBCH7/UBE2L3 (UBCH7) as indicated; ATP and ubiquitin were added (+) in lanes as noted. Immunoblots are for total ubiquitin. Brackets identify high mass conjugates in stacking gel. A) Ubiquitinylation with WT ubiquitin. Left: total ubiquitinylation; right, after Ni-NTA agarose purification. First 4 lanes in each blot contained His6-p27^Kip1; lanes 5–8 had no His6-p27^Kip1. B) p27^Kip1 was isolated as in (A), and ubiquitinylation was performed with K-only mutant ubiquitins: K11O, K29O, K63O, K48R or K48O. C) Quantification from 3 (K48R) or 4 (K48O) independent experiments.