A1 confocal microscope software

For imaging analysis, HEK293T cells grown on coverslips were co-transfected with GFP-tagged ANO1 vectors and pDsRed-Mem (Clontech Laboratories). Cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature (RT). After washing with PBS, cells were observed under a Nikon A1 confocal microscope. To quantify co-localization in merged images, at least 10 cells were randomly selected and analyzed. Pearson’s correlation coefficients were computed using Nikon A1 confocal microscope software. The r values are between 0 and 1. A value of 1 means that perfect co-localization occurs, while a value of 0 means that no co-localization is seen. Fluorescence intensity was measured using line scan-based analysis in Nikon A1 confocal microscope software. For BiFC, ANO1 and 14-3-3γ were cloned into pBiFC-VN173 and pBiFC-VC155 vectors (Addgene). HEK293T cells were co-transfected with cloned BiFC vectors in all possible pairwise combinations. The next steps were the same as those for imaging analysis, and Venus fluorescence signals were observed by confocal microscopy.