Reduced ORAI1-L194P expression and retention in the cytosol. (A and B) Cellular localization of ORAI1 in HEK293 cells overexpressing either ORAI1-WT-YFP, ORAI1-H134P-YFP, or ORAI1-L194P-CFP. (A) Representative confocal microscopy images. (B) Quantification of frequencies of cells with low ORAI1 expression levels (<5× background intensity). Scale bars are 50 µm. Data are from 200 to 400 cells per condition, from 5 independent experiments. P = 0.0004; 0.0003. (C) Flow cytometry analysis of cell surface ORAI1 protein expression in HEK293 cells. Cells were either transfected with ORAI1-WT-YFP, ORAI1-WT-CFP, ORAI1-H134P-YFP, ORAI1-L194P-CFP, or NT. Nonpermeabilized cells were stained with mouse anti-human ORAI1 (2C11) mAb against the second ECD. Representative flow cytometry plots. (D) Raw, non-normalized MFI of ORAI1 in all cells per condition. P = 0.821; 0.0066; 0.0049; 0.9995. Statistical significance was calculated using ordinary one-way ANOVA. ns, not significant, **P < 0.01, ***P < 0.001. NT, nontransfected.