Fig. 3.

Effect of PKC activation on the degradation rate of cell surface hOAT1. (A) hOAT1-expressing 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 2, 4, and 6 hours, respectively. Treated cells were lysed 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 PMA-treated and control at the same time point). (C) hOAT1-expressing 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. Treated cells were lysed and cell surface proteins were isolated using streptavidin-agarose beads, followed by IB with anti-E-cadherin antibody. (D) hOAT1-expressing cells were treated with or without PKC activator PMA at 37°C for 30 minutes. Treated cells were lysed, followed by IB with anti-Nedd4-2 antibody.