Fig. 4.

Effect of ubiquitin mutant Ub-K48R on the degradation rate of cell surface hOAT1. (A) hOAT1-expressing cells were transfected with wild-type ubiquitin (WT-Ub) or ubiquitin mutant (Ub-K48R). Transfected cells were biotinylated with membrane impermeable biotinylation reagent sulfo-NHS-SS-biotin. Labeled cells were then treated with or without PKC activator PMA at 37°C for 4 or 8 hours, respectively. Cells were lysed afterward and cell surface proteins were isolated using streptavidin-agarose beads, followed by immunoblotting (IB) with anti-myc antibody. (B) Densitometry plot of results from (A) as well as from other independent experiments. The values are mean ± S.E. (n = 3). *P < 0.05 (between WT-Ub- and Ub-K48R-transfected cells at the same time point). (C) hOAT1-expressing cells were transfected with WT-Ub or Ub-K48R. Transfected cells were biotinylated with membrane impermeable biotinylation reagent sulfo-NHS-SS-biotin. Labeled cells were then treated with or without PKC activator PMA at 37°C for 6 hours. Cells were lysed afterward and cell surface proteins were isolated using streptavidin-agarose beads, followed by IB with anti-E-cadherin antibody.