(A) COS cells were cotransfected with ROMK and ARH (+ARH) or empty vector (–ARH). Shown are typical results of surface ROMK, as detected in Western blot with ROMK antibodies, following cell surface biotinylation and neutravidin recovery, 72 hours after transfection. Surface is compared with total cell input of ROMK. Actin is not labeled by biotin, confirming biotinylation is specific for surface proteins. (B) Quantification of channel cell surface expression in the absence (black circles) and presence of exogenous ARH (red squares) at posttransfection times indicated (n = 3). ROMK surface expression is normalized to the average at time 0. The inset shows a Western blot of ARH in ARH-transfected cells over the time course. *P < 0.01. (C) Direct measurements of ROMK endocytosis in COS cells in the absence and presence of ARH (48 hours after transfection). Surface channels were labeled with sulfo-NHS-SS-biotin in the cold and then incubated at 37°C to permit endocytosis for variable times (0–7.5 minutes). Biotin remaining at the cell surface was cleaved with the reducing agent, MesNa, and internalized (biotinylated) proteins were recovered with neutravidin-conjugated beads and detected along with the total surface pool in immunoblots, using an anti-ROMK antibody. No proteins were captured on neutravidin beads without prior biotinylation (second lane). (D) Quantification of internalized channel relative to the surface pool at each time point by densitometry (mean ± SEM; n = 3). *P < 0.01. (E) Rates of ROMK endocytosis in the absence and presence of exogenous ARH. *P < 0.01.