Figure 2. Orai1 and Orai3 mediate the bulk of store-operated Ca2+ entry (SOCE) in A20 B lymphoblasts.

(A) Cartoon schematic of the two gRNA CRISPR strategies we used to excise mouse Orai1 and Orai3 genes. (B–D) Quantitative RT-PCR of (B) Orai1, (C) Orai2, and (D) Orai3 mRNA in A20 Orai CRISPR clones (n=3 biological replicates). (E) Measurement of SOCE with Fura2 upon store depletion with 2 µM thapsigargin in 0 mM Ca²⁺ followed by re-addition of 2 mM Ca²⁺ to the external bath solution. (F) Quantification of peak SOCE in (E) (from left to right n=99, 100, 89, and 98 cells; Kruskal-Wallis test with multiple comparisons to WT A20). (G–J) Representative Ca²⁺ oscillation traces from 5 cells/condition measured with Fura2 upon stimulation with 10 μg/mL anti-IgG antibodies at 60 s (indicated by arrows) in the presence of 2 mM external Ca²⁺. (K) Quantification of total oscillations in 9 min from (G–J) (from left to right n=76, 79, 79, and 78 cells; Kruskal-Wallis test with multiple comparisons to WT A20). All scatter plots are presented as mean ± SEM. For all figures, *p<0.05; **p<0.01; ****p<0.0001; ns, not significant.