Figure 2.
Reduced expression of the ORAI1 mutant protein at the cell surface. (A) Cellular localization of ORAI1 in HEK293 cells overexpressing either ORAI1-WT-YFP, ORAI1-H134P-YFP, or ORAI1-L194P-CFP. Representative confocal microscopy images (left), and quantification of frequencies of cells having PM or intracellular ORAI1 expression patterns (right). Scale bars are 5 µm. Data are from 200 to 400 cells per condition and 5 independent experiments. Cells with low expression (<5× background intensity) were excluded from the analysis. P ≤ 0.0001; <0.0001; <0.0001; 0.0777; >0.9999. (B–E) Flow cytometry analysis of cell surface ORAI1 protein expression in HEK293 cells. Nonpermeabilized cells were stained with a mouse anti-human ORAI1 (29A2) mAb that recognizes amino acids 196–234 in the second extracellular loop of hORAI1 (19). (B) Cells were transfected with either ORAI1-WT-YFP, ORAI1-H134P-YFP, ORAI1-WT-CFP, or ORAI1-L194P-CFP. Frequency of cells expressing ORAI1 at the cell surface based on the gating strategy shown in Fig. S2 C (P = 0.9233; <0.0001). (C–E) Representative flow cytometry plots (left) and quantification of MFI of ORAI1 mutants normalized to the corresponding ORAI1-WT control (right). Data are from three independent experiments. (C) Cells were transfected with either ORAI1-WT-YFP, ORAI1-H134P-YFP, or nontransfected cells. P = 0.0299; <0.0001; <0.0001. (D) Cells were transfected with either ORAI1-WT-CFP, ORAI1-L194P-CFP, or nontransfected cells. P = <0.0001; <0.0001; 0.5304. (E) Cells were cotransfected with either ORAI1-WT-YFP and ORAI1-WT-CFP, ORAI1-H134P-YFP and ORAI1-L194P-CFP, or nontransfected cells. P = 0.0041; <0.0001; 0.0002. Statistical significance was calculated using two-way ANOVA (A) and ordinary one-way ANOVA (B–E). ns, not significant, *P <0.05, **P <0.01, ***P <0.001.

Reduced expression of the ORAI1 mutant protein at the cell surface. (A) Cellular localization of ORAI1 in HEK293 cells overexpressing either ORAI1-WT-YFP, ORAI1-H134P-YFP, or ORAI1-L194P-CFP. Representative confocal microscopy images (left), and quantification of frequencies of cells having PM or intracellular ORAI1 expression patterns (right). Scale bars are 5 µm. Data are from 200 to 400 cells per condition and 5 independent experiments. Cells with low expression (<5× background intensity) were excluded from the analysis. P ≤ 0.0001; <0.0001; <0.0001; 0.0777; >0.9999. (B–E) Flow cytometry analysis of cell surface ORAI1 protein expression in HEK293 cells. Nonpermeabilized cells were stained with a mouse anti-human ORAI1 (29A2) mAb that recognizes amino acids 196–234 in the second extracellular loop of hORAI1 (19). (B) Cells were transfected with either ORAI1-WT-YFP, ORAI1-H134P-YFP, ORAI1-WT-CFP, or ORAI1-L194P-CFP. Frequency of cells expressing ORAI1 at the cell surface based on the gating strategy shown in Fig. S2 C (P = 0.9233; <0.0001). (C–E) Representative flow cytometry plots (left) and quantification of MFI of ORAI1 mutants normalized to the corresponding ORAI1-WT control (right). Data are from three independent experiments. (C) Cells were transfected with either ORAI1-WT-YFP, ORAI1-H134P-YFP, or nontransfected cells. P = 0.0299; <0.0001; <0.0001. (D) Cells were transfected with either ORAI1-WT-CFP, ORAI1-L194P-CFP, or nontransfected cells. P = <0.0001; <0.0001; 0.5304. (E) Cells were cotransfected with either ORAI1-WT-YFP and ORAI1-WT-CFP, ORAI1-H134P-YFP and ORAI1-L194P-CFP, or nontransfected cells. P = 0.0041; <0.0001; 0.0002. Statistical significance was calculated using two-way ANOVA (A) and ordinary one-way ANOVA (B–E). ns, not significant, *P <0.05, **P <0.01, ***P <0.001.

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