Figure 7. Ectopic expression of α-SNAP can restore defects in Napahyh/hyh CD4 T cells.
(A) Average cytosolic calcium levels, measured using FURA 2AM, in scr (black) and α-SNAP RNAi (red)-treated cells stimulated with anti-CD3 antibody to measure SOCE. (n = 3 with ~50 to 100 cells per experiment). (B) Quantitative PCR to estimate the expression of key effector cytokines in scr (black) and α-SNAP RNAi (red)-treated Th0 cells. (n = 2 repeats; samples from 3 repeats of RNAi). (C) Representative FACS profiles showing intracellular IL-2 staining in WT and Napahyh/hyh CD4 T cells reconstituted with EV, WT or M105I α-SNAP. (n = 3). (D) Average cytosolic calcium levels, measured using FURA 2AM, in anti-CD3- stimulated WT and Napahyh/hyh CD4 T cells expressing empty vector (EV), WT or M105I α-SNAP. (n = 2 with ~50 to 200 cells each). (E) Western blot showing in vitro binding of WT and M105I α-SNAP to Stim1 and Orai1. (n = 2). (F) Confocal images of HEK293 cells expressing WT or M105I α-SNAP and stained with anti-α-SNAP antibody and DAPI (Scale bar 10 μm). (n = 2; 5 to 6 cells/ per group/ experiment). (G) TIRF images of store-depleted HEK 293 cells co-expressing CFP-Stim1 and YFP-tagged WT or M105I α-SNAP. (Scale bar 10 μm). (n = 2 with 5 to 6 cells/ per group/ experiment).