Fig 1: The V1 and VO domains of the V-ATPase are less assembled in the kidneys of Dmxl1fl/fl, B1-Cre+ mice. (A) Western blots of subcellular fractionation experiments showing decreased membrane (mem) accumulation of the cytosolic V1-associated B1 subunit of the V-ATPase in Cre+ mice relative to Cre− mice (top). The membrane-bound VO-associated a4 subunit is only detected in the membrane fraction, as expected, confirming the purity of the preparation (bottom). (B) Quantification of the membrane: cytosol ratio of the cytosolic V1-associated B1 subunit of the V-ATPase. (C) Immunofluorescence images taken from the outer medulla of kidneys from Cre− (top) and Cre+ (bottom) mice after performing a proximity ligation assay (PLA, spots, center) using specific antibodies against the B1 and a4 V-ATPase and post-staining with a specific antibody against AE1 (left). Association of B1 with a4 is represented by the increased number of spots in A-type intercalated cells (A-ICs) from Cre− mice. Basolateral AE1 staining was used to identify A-ICs (indicated with arrowheads). Nuclei are labeled with DAPI (right). Scale bars = 10 µm. (D) Quantification of total area of PLA signal per A-IC shows a significant decrease of B1/a4 association in Cre+ mice. Values for 25 cells were averaged per animal. Data were analyzed by t-test with values reported as means ± SEM with P-values ≤ .05 considered significant, with * denoting a P-value ≤ .05, and ** ≤ .001.