Localization and vasopressin regulation of the Na+-K+-2Cl- cotransporter in the distal colonic epithelium

Figure 1 Expression and spatial distribution of Na⁺-K⁺-2Cl^- cotransporter in the mouse colon and kidney. RT-PCR identified mRNA transcripts for NKCC2 (1161 bp) in the mouse distal colon mucosa (lane 1) and kidney (lane 3) (A). In control experiments, no band was detected using non reverse transcribed RNA (lane 5) or by omitting cDNA (lane 6). The expression of β-actin is also shown as an internal control (lanes 2 and 4). Localization of the NKCC1 protein in the basolateral membrane along lower crypt epithelium (B) (arrowhead, the left inset is enlarged image of white rectangle) and NKCC2 in the apical membrane along the surface epithelium (C) (arrowhead, the left inset is enlarged image of white rectangle n = 3 mice) were clearly observed. Preadsorption of NKCC1 and NKCC2 antibodies with their corresponding blocking peptides (+ BP) resulted in no immunoreactivity (D, H). NKCC2 expression in the apical membrane of TAL cells in the kidney served as a positive control (F). Primary NKCC2 antibody was omitted also as a negative control (G). NKCC labeling with T4 antibody at the surface and crypt of the colonic epithelium (arrowhead: apical arrow: basolateral) (E) Higher magnification of surface epithelium (I). Scale bar = 20 μm; NC: Negative control; NKCC2: Na⁺-K⁺-2Cl^- cotransporter.