Figure 1: Segment a of ANO1 is critical for the expression of ANO1 at the plasma membrane.

(a) Schematic representation of ANO1(abc) isoform and its deletion mutant. Black boxes: transmembrane domains; hatched box: segment a; diagonal striped box: segment b, gray box: segment c. (b) Cell surface biotinylation showed that segment a is critical for the surface expression of ANO1 in GFP-ANO1(abc)- or GFP-ANO1(abc)-Δa-transfected HEK293T cells. Western blot images were cropped for clarification. Full blot images were included in supplementary information. (c) Co-localization of GFP-ANO1(abc) or GFP-ANO1(abc)-Δa with DsRed-Mem in HEK293T cells. Scale bars, 10 μm. The presence and absence of co-localized GFP signals and membrane markers are shown as an arrow and arrowhead, respectively, in the enlarged images. (d) Pearson’s correlation coefficients denoting covariance of fluorescence signals were calculated using Nikon software as in (c). Data are expressed as mean ± SEM; ***P < 0.001. (e) Representative traces of whole-cell recordings of COS7 cells transfected with GFP-Con, GFP-ANO1(abc), or GFP-ANO1(abc)-Δa. (f) Summary bar graph showing normalized current density as in (e) at +80 mV. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01.