Fig. 2

AC8 mediates ORAI1 CDI through physical interaction with ORAI1. a–c Representative currents from ORAI1-KO cells co-expressing eYFP-STIM1 with ORAI1-CFP and co-transfected with a control non-targeting siRNA or b siRNA against AC8. c The extent of ORAI1 CDI from (a) and (b) represented as current remaining at 146 ms. Each data point represents mean ± SEM. d Western blot of AC8 in cells transfected with control non-targeting siRNA and AC8 siRNA. e Densitometry of AC8/GAPDH proteins from three independent experiments and shown as boxplots representing the 25th to 75th percentile range, mean, and median. f–i Representative currents from ORAI1-KO cells co-expressing eYFP-STIM1 with ORAI1-CFP and co-transfected with f an empty vector control, g AC8 cDNA, or h AC8M1 cDNA. i The extent of ORAI1 CDI from (f), (g), and (h) represented as current remaining at 146 ms. Each data point represents mean ± SEM. j Fluorescence image of ORAI1-KO cells co-expressing ORAI1-CFP (red) and eYFP-tagged versions (green) of either AC8 (top) or AC8M1 (bottom). Scale bar: 10 µm (k, l). k Pull-down assay of AC8 with ORAI1-CFP and ORAI1β-CFP expressed in ORAI1-KO cells. l Quantification of AC8 pull-down data from five independent experiments and represented as boxplots showing the 25th to 75th percentile range, mean, and median. m e-FRET data of interactions between CFP-AC8 and either eYFP- ORAI1 or eYFP-ORAI1β before and after addition of carbachol (Cch, 500 µM) with each point representing mean ± SEM. n YFP/CFP fluorescence ratios from recordings in (m) represented as boxplots showing the 25th to 75th percentile range, mean, and median. o, p Representative fluorescence and FRET images of cells expressing o CFP-AC8 and eYFP- ORAI1, or p CFP-AC8 and eYFP-ORAI1β. Scale bar: 10 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-tailed Student’s t test was used for (c, e, l, m, n), and one-way ANOVA for (i)