Figure 4.

UbcH7 is a substrate of the ubiquitin proteasome pathway: (A) HeLa cells were treated for 4 h with cycloheximide in the presence or absence of MG132 as indicated (top). Levels of UbcH7 were normalized to E1 (bottom). (B) Ubiquitination reactions were performed with ¹²⁵I-UbcH7, reticulocyte lysate, Ubc4, and ubiquitin as indicated. Bands for monoubiquitinated (bottom panel, short exposure) and polyubiquitinated (top panel, long exposure) UbcH7 are noted. A nonspecific band is indicated by an asterisk. As noted at the top of the gel in lane 2, some low background level of UbcH7 oligomerization or ubiquitination is occurring in the presence of lysate and Ubc4. (C) ¹²⁵I-UbcH7 was added to reticulocyte lysates containing ATP, Ubc4, and MG132 as indicated. Degradation was calculated by comparing the TCA-soluble counts to total radioactivity after 2 h. (D) HeLa cells synchronized using HU as in Figure 1A were lysed, and the resulting extracts were used for degradation of ¹²⁵I-UbcH7 in the presence of Ubc4, ATP, and ubiquitin. The data shown are the average of five experiments with independent extracts. *p < 0.05 S versus G2/M.