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doa4Δ pep4Δ cells accumulate huge amounts of Ub conjugates. MOB100 (pep4Δ) and SD21 (doa4Δ pep4Δ) cells transformed with YEp96fF (2μm URA3 FUR4 CUP1-UB) were grown and induced for Ub with CuSO4 as described in the legend to Fig. 1. They were treated with CHX for the times indicated. Membrane-enriched fractions (P13 and P100) were prepared. Aliquots were analyzed by Western immunoblotting for Ub conjugates, Pma1p, and Pep12p. The position of size standards are indicated on the left.
