Fig. 4

Ca²⁺-CaM dissociates the SOAR–Orai1 complex. a, b Resting FRET was measured between Orai1-CFP and SOAR (WT or mutant)-YFP on PM of HEK Orai1-CFP stable cells. The cells were in 0 mM extracellular calcium Ringer’s buffer. Scale bars are 10 μm. c Confocal images of live HEK293T cells transfected with SOAR-GFP (WT or mutant) and Cherry-Orai1. Scale bars are 10 μm. d Western blot analysis of co-immunoprecipitated SOAR-GFP (WT or mutant) with Myc-Orai1. e Ca²⁺ influx in HEK293T cells co-expressing Cherry-Orai1 and SOAR-YFP WT or mutant. f Western blot analysis of competitive binding of the SOAR–Orai1 complex in the presence or absence of Ca²⁺-CaM. g Steady-state FRET between Orai1-CFP and YFP-SOAR co-expressed with CaM or CaM-4EF mutant on PM of HEK293T cells. FRET values were measured after 2 mM extracellular Ca²⁺ was added. h Steady-state FRET between Orai1-CFP and YFP-SOAR on the PM of HEK293T cells that were pretreated with 30 μM W-7 or DMSO control for 30 min. FRET values were measured after 2 mM extracellular Ca²⁺ was added. Numbers of cells that were analyzed are indicated. *P < 0.05, **P < 0.01 (unpaired Student’s t-test). Error bars denote SEM