Figure 2. Ca²⁺ influx measurements show that Drosophila Orai constructs function as CRAC channels when co-expressed with STIM.

HEK293 cells were transfected with Orai and/or STIM as indicated (also see Materials and methods). Cytosolic [Ca²⁺] levels were detected using the genetically-encoded fluorescent Ca²⁺ indicator GCaMP6s (Chen et al., 2013); data are plotted as the change in fluorescence intensity relative to the initial value (ΔF/F0) versus time. Thapsigargin (TG), which is used to deplete ER calcium stores, and 2 mM CaCl₂ were added at the indicated times (arrows). Ca²⁺ influx above the background level (vector alone) was observed for the co-expression of STIM with Orai_cryst-RR or wild-type Orai, but not for the constitutively closed K163W mutant or for Orai or STIM alone. Signal from endogenous CRAC channels is apparent from the vector control (empty expression vector). Standard error, derived from three independent measurements, is shown.