Figure 6. Orai1 is a regulator of nuclear factor of activated T cells (NFAT) activation in naïve and activated B cells.

(A) Representative Imagestream images following intracellular staining for NFAT1 and DAPI in naïve B cells from Mb1-Cre^/+ mice before and after 20 μg/mL anti-IgM stimulation for 15 min. Merge image indicates similarity score co-localization between NFAT1/DAPI. (B) Histograms of NFAT1/DAPI similarity scores before (black trace) and after (red trace) anti-IgM stimulation in naïve B cells from Mb1-Cre^/+ and Orai1^fl/fl Mb1-Cre^/+ mice. (C) Quantification of similarity scores following anti-IgM stimulation in (B) (n=3 biological replicates for each; unpaired T-test). (D) Western blot analysis of NFAT1 and α-tubulin in naïve B cells isolated from Mb1-Cre^/+ and Orai1^fl/fl Mb1-Cre^/+ mice. B cells were left unstimulated or treated with 2 µM thapsigargin (Tg) for 15 min before harvesting. (E) Quantification of NFAT1 dephosphorylation in (D) (n=4 biological replicates for each; Mann-Whitney test). (F) Western blot analysis of NFAT1 and α-tubulin in B cells stimulated for 48 hr with anti-IgM +anti-CD40. (G) Quantification of NFAT1 dephosphorylation in (F) (n=3 biological replicates for each; Mann-Whitney test). All scatter plots are presented as mean ± SEM. For all figures, *p<0.05; **p<0.01; ****p<0.0001.

Figure 6—figure supplement 1. Nuclear factor of activated T cells (NFAT) activation in primary Orai1/Orai3-deficient B cells.

(A) Western blot analysis of NFAT1, NFAT2, and α-tubulin in naïve B cells isolated from Mb1-Cre^/+ and Orai1^fl/fl Mb1-Cre^/+ mice. B cells were either unstimulated or treated with 20 μg/mL anti-IgM or 2 µM thapsigargin (Tg) for 15 min before harvesting. (B) Western blot analysis of NFAT1 in naïve B cells isolated from Mb1-Cre^/+, Orai1^fl/fl Mb1-Cre^/+, Orai3^fl/fl Mb1-Cre^/+, and Orai1/Orai3^fl/fl Mb1-Cre^/+ mice following thapsigargin treatment. (C) Same as in (B) but with B cells stimulated for 48 hr with anti-IgM +anti-CD40.