Fig 2: Ano1 ± mouse caused reduction of basal and CCH-stimulated Isc. (A) Basal and CCH-stimulated Isc in the small intestine (jejunal) and colonic tissue of WT and Ano1 ± mice. Data are means ± S.E (n = 6).*P < 0.05. (B) Showing FSK-stimulated Isc response in WT and Ano1 ± mouse colonic mucosa. Data are means ± S.E (n = 4–6). (C) Representative western blot image of TMEM16A protein in different parts of wild type and Ano1 ± enterocytes. (D) Quantitative analysis of the immunoblots was determined by ImageJ analysis. Data are means ± S.E of three independent experiments. (* denotes significant difference at p value < 0.05, NS, non significant at p < 0.05).

Fig 3: TMEM16A localizes to the apical membrane of mouse enterocytes and immunoprecipitates with NHERF1 in co-transfected HEK293T cells. (A) Representative western blot showing NHERF1 expression in mouse intestinal tissue as indicated. The histogram showing the densitometric quantification of NHERF1 protein expression, right to the immunoblot. This experiment was performed in triplicate. (B) Immunofluorescence demonstrated TMEM16A (green) and NHERF1 (red) in mouse colon, the yellow signal indicated co-localization of TMEM16A with NHERF1 (merged). Representative of two independent experiments. (C) TMEM16A co-immunoprecipitates with Flag-NHERF1 in co-transfected HEK293T cells. TMEM16A was detected only in the Co-IP complex from cell co-transfected with plasmids expressing both mouse Ano1 and Flag-Nherf1. (D)Depicted a reduced TMEM16A abundance in Co-IP complex from cell lysates with truncated (delC4) Ano1 compared to the result obtained with the control (full length). Representative blot of three independent experiments for (C) and (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: Functional involvement of NHERF1 in TMEM16A mediated Cl− secretion. (A) NHERF1 was co-immunoprecipitated with TMEM16A endogenously from T84 cell lysates. No signal was detected using protein-G-conjugated beads alone (IP control). Representation of three independent experiments. (B) Western blot showing NHERF1 expression in wild type T84 (T84WT), vector control (T84VC) and NHERF1 knocked down cells (T84NF1KD736). (C) Histogram showing densitometric quantification of NHERF1 expression, right to the immunoblot. Result represents means ± S.E (n = 3). (D) Representative traces showing the response of T84NF1KD736 cells to CCH-stimulated Isc. Inset showing summarized data from six independent experiments. Data are means ± S.E. (E) Effect of FSK pre-stimulation on CCH-stimulated Isc in WT and NHERF1KD T84 cell monolayers. Monolayers grown on filters were exposed to FSK (10 μM) and then stimulated with serosal CCH (100 μM). Data are mean ± SEM from 4 to 6 monolayers of each condition.