Figure 2
Colocalization and coimmunoprecipitation of ARH and ROMK in the rat kidney.
Immunofluorescent staining of (A) ROMK with a chicken anti-ROMK antibody and (B) ARH with a goat anti-ARH antibody (C) and colocalization of ARH (green) and ROMK (red) in a rat kidney collecting duct, using appropriate, specific Alexa Fluor–conjugated secondary antibodies. Arrowheads indicate subapical or perinuclear labeling in principal cells; asterisks indicate intercalated cells. Scale bar: 8.5 μm. (D) Low-power confocal microscope image of ARH localization in cortex of rat kidney. ARH can be detected in cortical collecting ducts (CCDs), TALs, and distal tubules (DTs), but little or no labeling is detected in proximal tubules (asterisks). Collecting ducts were identified based on double labeling with antibody to AQP2 raised in chicken and other segments identified based on morphological criteria. Scale bar: 8 μm. (E) Immunoprecipitation analysis of ROMK channel with ARH from whole rat kidney extracts. (F) Immunoprecipitation of HA epitope–tagged ROMK or mutant ROMK, lacking the NPNF motif, with myc-tagged ARH from COS-7 cells transfected with indicated (+) cDNAs. Only WT ROMK was immunoprecipitated with ARH. (G) Quantification of IP results. n = 3; *P < 0.001.
