Fig. 5

Ca²⁺-CaM dissociates the STIM1–Orai1 complex. a Confocal images of live HEK293T cells transfected with STIM1-GFP (WT or mutant) and Cherry-Orai1 before and after 1 μM thapsigargin (TG) stimulation. Scale bars are 10 μm. b, c FRET was measured between Orai1-CFP and STIM1-YFP (WT or mutant). TG (1 μM) was used to induce Ca²⁺ depletion. The same cell before and after TG stimulation is shown. Scale bars are 10 μm. d Ca²⁺ influx in HEK293T cells co-expressing Cherry-Orai1 and STIM1-YFP WT or mutant. e Western blot analysis of co-immunoprecipitated STIM1-GFP (WT or mutant) with Myc-Orai1. TG (1 μM) was used to induce Ca²⁺ depletion before lysis. f Western blot analysis of competitive binding of Ca²⁺-CaM protein with STIM1–Orai1 complex in HEK293T cells. Ionomycin (3 μM) was used to induce Ca²⁺ depletion before lysis. g Steady-state FRET between Orai1-CFP and STIM1-YFP that were co-expressed with CaM or CaM-4EF mutant after 3 μM ionomycin treatment in HEK293T cells. h Steady-state FRET between Orai1-CFP and STIM1-YFP in HEK293T cells that were pretreated with 30 μM W-7 or DMSO control for 30 min after 3 μM ionomycin treatment. Numbers of cells analyzed are indicated. ***P < 0.001 (unpaired Student’s t-test). Error bars denote SEM