Fig. 2. H2 site mutants of STIM1 CaM-binding domain show impaired Orai1 activation in HEKO1 STIM1^-/- cells. (A), Anti-Orai1 Western blot of HEK293 wild type and HEK293 cells stably overexpressing Orai1 (HEKO1). (B), Anti-STIM1 Western blot of HEKO1 and STIM1 knock-out HEKO1 (HEKO1 STIM1^-/-) cells. (C), Representative FLIPR traces of calcium entry after thapsigargin mediated store-depletion in HEKO1 (black) and HEKO1 STIM1^-/- (green) cells. 1 µM thapsigargin (Tg) and 2 mM CaCl₂ (Ca²⁺) administrations are indicated. (D), Representative FLIPR traces of SOCE in HEKO1 STIM1^-/- cells transiently transfected with mCherry (Control, red), mCherry-STIM1 (STIM1, black) or mCherry-STIM1-L390A-F391A (STIM1 H2AA, blue). (E), Quantified area under the curve (AUC) and (F), slope of the SOCE traces after extracellular Ca²⁺ application in HEKO1 STIM1^-/- cells transiently transfected with control (red), STIM1 (black) or STIM1 H2AA (blue). The data was normalized by setting the AUC and slope of the Ca²⁺ entry traces to 100 (n = 15; mean ± standard deviation). (G), average current density (CD) of the I_CRAC recordings (STIM1, n = 9; STIM1 H2AA, n = 6; mean - SEM) from HEKO1 STIM1^-/- cells. (H), average current-voltage (I-V) curves of the I_CRAC recordings at t = 2 min (STIM1, n = 9; STIM1 H2AA, n = 6). (I), quantified average CD values at t = 2 min (STIM1, n = 9; STIM1 H2AA, n = 6; mean ± SEM) from HEKO1 STIM1^-/- cells transiently overexpressing STIM1 (black) or STIM1 H2AA (blue). (J), Representative FLIPR traces of SOCE in HEKO1 STIM1^-/- cells transiently transfected with mCherry (Control, red), mCherry-STIM2 (STIM2, black) or mCherry-STIM2-V394A-F395A (STIM2 H2AA, blue). (K), Quantified AUC and (L), slope of the SOCE traces after extracellular Ca²⁺ application in HEKO1 STIM1^-/- cells transiently overexpressing control (red), STIM2 (black) or STIM2 H2AA (blue). The data was normalized by setting the AUC and slope of the Ca²⁺ entry traces to 100 (n = 15; mean ± standard deviation). p value (p) of the WT or mutant STIM compared to the mcherry control group is indicated above the respective bar as non-significant (n.s.) for p > 0.05 or as "***" for p <= 0.001.