[PubMed] [CrossRef] [Google Scholar] 21. accumulates in the cytoplasm and the subapical and apical membrane. In addition, the mutant transporter targets some of wild-type transporter to the apical membrane. Disruption of epithelial integrity is likely to affect key physiological processes. One of these processes is usually saliva production, which is usually mediated by acinar cells of the parotid, submandibular, and sublingual glands. In normal conditions, fluid secretion originates at the base of the gland and is mediated by Cl? movement through the epithelial cells, via basolateral NKCC1-mediated transport and apical Cl? channels. Cl? is later exchanged for bicarbonate in the ducts (37). It is known that disruption of NKCC1 (12) or CFTR (1) function in mice results in decreased saliva secretion. Here we show that NKCC1 localizes to the subapical and apical membrane of salivary epithelial cells in a mouse model of the patients mutation. Despite the mistargeting of some NKCC1 protein to the apical membrane, stimulated saliva secretion was not affected in the NKCC1 mutant mice. MATERIALS AND METHODS Reagents. Prolong Gold Antifade reagent with DAPI was obtained from Life Technologies (Grand Island, T863 NY). Matrigel was obtained from Corning, and ActinRed and ActinGreen ready probes were purchased from Invitrogen (Carlsbad, CA). Streptavidin agarose resin was purchased from Thermo Scientific (Waltham, MA). The following antibodies were used as primary antibodies: NKCC1 (T4) from the University of Iowa Developmental Studies Hybridoma Lender (DSHB: T4 monoclonal, 1:1,000); PDXL/Gp135 (DHSB: Podocalyxin catalog no. 3F2D8 monoclonal, 1:500) ; NKCC1 (Abcam, catalog no. ab59791, 1:2,000); zonula occludens-1 (ZO-1; DSHB: R26.4 monoclonal, 1:200); green T863 fluorescent protein (GFP; Vanderbilt VAPR Core, 1C9A5 monoclonal; 1:1,000); Alexa-Fluor-488 tagged anti-GFP (ThermoFisher: catalog no. A-21311; 1:200); Ezrin (Millipore, Clone T863 4A5, catalog no. MAB3822-C; 1:200); anti-tdTomato (ThermoFisher, Clone RF5R; catalog no. MA5-15257, 1:200), -actin (Sigma, AC-74 monoclonal, catalog no. A2228; 1:5,000), and anti c-Myc (ThermoFisher, clone 9E10; catalog no. MA1-980, 1:2,000). cDNA clones. The enhanced GFP (EGFP) and tdTomato (TdT) open reading frames were cloned at the extreme amino terminus of the 3.7 kb mouse NKCC1 cDNA. In the process, the first nine amino acid residues of Bmp6 NKCC1 were eliminated. Prior experiments performed in oocytes exhibited no functional differences between wild-type NKCC1 and EGFP-NKCC1 constructs (17). The mouse cDNA carrying the T863 DFX mutation was previously described (9). Epitope-tagged NKCC1-WT and NKCC1-DFX constructs were subcloned into pCDNA3 plasmid. To create the mutant NKCC1-G345R mutant, we first subcloned a 363 bp for 16 h. Fourteen fractions of ~800 l each were then collected from the top of the gradient. Aliquots (40 l) of each fraction were mixed with 40 l sample buffer made T863 up of 500 mM of DTT and denatured at 75C for 20 min. Fractions were resolved on 10% polyacrylamide gel electrophoresis and analyzed by Western blotting. Live cell imaging. MDCKWT and MDCKDFX were cultured on MatTek Glass Bottom Microwell dishes until they reached 100% confluency. Cells were then washed twice with HBSS2+ and incubated in HBSS2+ made up of 1 g/ml of PureBlu Hoechst 33342 (Bio-Rad) and 100 M of ER-Tracker Red (BODIPY TR, ThermoFisher) for 30 min at 37C. Cells were then washed twice with HBSS2+, incubated in HBSS2+ made up of 2.5% FBS, and imaged. All images were captured live on a Zeiss LSM 880 laser scanning confocal microscope. Samples were scanned using a 63 oil objective. The images were exported as TIFF files using Zeiss ZEN Lite 2012 software. Immunofluorescence. MDCK cells expressing epitope-tagged NKCC1-WT and NKCC1-DFX were cultured on glass coverslips until they reached 100% confluency. Cells were fixed with cold (?20C) methanol, washed, and mounted on.