Fig. 4.
Two STIM1-C proteins bind to Orai1 complexes. (A) Upper shows a representative confocal image captured in the middle focal plane of live HEK293 cells cotransfected with STIM1-C-EGFP and Orai1-mKO. Bottom shows colocalization of these two proteins at the membrane under TIRF illumination again in live cells. (Scale bars, 10 μm.) (B) Representative fluorescence steps in TIRF microscopy for HEK293 cells transfected with STIM1-C-EGFP and no exogenous Orai1. Puncta bleached either in two steps (Upper) or one step (Lower). (C) Comparison of one- and two-step bleaches in subpopulations of cells from the same dish expressing low (253 ± 19 arbitrary units; n = 3) or higher (405 ± 41; n = 3) amounts of STIM1-C-EGFP. The higher expressers had significantly more one-step bleach events occurring per μm2 (P < 0.05) but no change in two-step events.