Store depletion in primary salivary gland cells and STIM1 Orai1 localization in HeLa cells. (A) The transient application of CPA (30 µM) depletes ER Ca²⁺ stores as indicated by the rise in cytosolic Ca²⁺ (blue) which activates the Cl⁻ current (red). Following the washout of CPA, the application of carbachol (CCh, 10 µM) fails to elicit a response, indicating the effective depletion of the ER stores (n = 4–8). (B and C) Kinetics of intracellular Ca²⁺ elevation and Cl⁻ current development during tunneling compared to SOCE (n = 26 (B) and 9 (C); unpaired t test). (D) Enlarged confocal images show the colocalization of STIM1 and Orai1 at the PM focal plane. (E and F) Plots from a virtual line scan drawn across clusters in the PM plane (E, arrow in D) and in the z axis (F).