Fig. 7

Effect of 2NQ(6-11) mutations on EFSAM Ca²⁺ sensing. a–b Confocal micrographs of GFP-STIM1-2NQ(6-11) (6-11; a) and GFP-STIM1 (WT; b) at rest (2 mM Ca²⁺; left panels), during a 10-min exposure to Ca²⁺-free solution (0 mM Ca²⁺; middle panels), and following store depletion with 1 μM TG (right panels). Scale bars, 5 μm. c Single-cell [Ca²⁺]_i measurements in HeLa cells expressing mCherry-ORAI1 and wild-type GFP-STIM1 (WT; n = 220), GFP-STIM1-2NQ(6-11) (6-11; n = 258), or GFP-STIM1-2NQ (2NQ; n = 193), or in non-transfected HeLa cells (HeLa; n = 220). Cells were exposed to solutions containing varied concentrations of CaCl₂ or 1 μM thapsigargin (TG) as indicated. d ITC analysis of EFSAM-2NQ(6-11). Top panel, heat changes measured by injecting 1 μl aliquots of 10 mM Ca²⁺ into a sample cell containing initially 100 μM protein. Bottom panel, integrated binding isotherm as a function of molar ratio (Ca²⁺: protein) after subtracting heats of dilution. Total Ca²⁺ at 70 min is the amount added to the ITC cell up to that point, corrected for the increase in volume, and free Ca²⁺ is calculated as total Ca²⁺ minus bound Ca²⁺ assuming five sites are occupied in EFSAM. e Western blot showing crosslinking of STIM1(A230C)-2NQ(6-11) in cellular membranes incubated at the specified Ca²⁺ concentrations. The black arrow marks the STIM1 monomer band, and the red arrow marks the dimer band. The uncropped blot is shown in Supplementary Fig. 12. f STIM-STIM dimer formation for STIM1(A230C)-2NQ(6-11) at each Ca²⁺ concentration is compared to the corresponding dimer formation for STIM1(A230C). Data from three independent experiments are plotted as mean ± SEM