Fig. 5. (A) Effect of Ubc6p overexpression on mammalian ERAD. The turnover of TCR and F508CFTR was determined in pulsechase experiments with or without expression of wild-type and mutant Ubc6. The calculated half-life of both substrates is summarized in the plot. The turnover was measured upon co-expression of either mmUbc6 or hsUbc6e as indicated. Of each conjugating enzyme a wild-type version (grey bars) or an active site mutant (black bars) was used. As a control, an unrelated protein (GFP; white bars) was expressed. For TCR, data indicated are mean±s.e.m. of three experiments derived from linear regressions of semi-logarithmic transformations of decay kinetics. For F508 CFTR, data are from two representative experiments. Below the plot, representative autoradiograms are shown. (B) Cells stably expressing an unstable cytosolic protein GFP^u were transfected with wild-type hsUbc6e or mutant hsUbc6e-C91S, and GFP fluorescence was determined by FACS analysis 2 days later.