Halide-sensitive YFP assay demonstrates Ca⁺²-dependent anion conductance in HEK293 cells expressing TMEM16B. A , Representative traces show fluorescence (arbitrary units) in HEK293 cells transfected with YFP-I152L (dashed line) or with YFP-I152L and TMEM16B (solid line). Fluorescence quenching was induced by isotonic application of 20 mm I⁻ (gray arrow) and 1 μm ionomycin (black arrow). B , Summary of I⁻ influx-mediated maximal fluorescence quenching (mean ± SEM). Gray boxes show maximal quenching after the addition of I⁻, black boxes demonstrate maximal quenching after ionomycin treatment. Addition of ionomycin induced significant fluorescence quenching in all YFP- I152L-transfected cells (*p < 0.0001; paired Student's t test) (n = 51). Ionomycin-mediated fluorescence quenching was significantly enhanced when YFP-I152L was cotransfected with TMEM16B (**p < 0.001; ANOVA) (n = 61). Pretreatment with DIDS had no effect on ionomycin-induced I⁻ influx in cells expressing TMEM16B (n = 31).