Localization of SERCA2b and mCh-STIM1. (A) HeLa cells transfected with mCh-STIM1 and stained using a SERCA2b antibody. AiryScan images were taken before (Ctr) and after store depletion (Tg). Control images are acquired at a focal plane located in the middle of the cell and store-depleted images at the PM plane, where the STIM1 clusters localize. At rest, STIM1 and SERCA2b colocalize in the ER cisternae, while after store depletion they separate from each other at the PM focal place. (B) Pearson’s correlation coefficient (PCC) obtained before (Ctr) and after store depletion (Tg) between endogenous SERCA2b and overexpressed mCh-STIM1. Colocalization at rest was measured in the middle of the cell and at the PM plane after store depletion (n = 9–11; unpaired t test). (C) Image and line scan of STIM1 clusters on the side of the cell depicting its isolation from SERCA2b. (D) Cartoon illustrating the localization of STIM1 (red) and SERCA (green) after store depletion. The positions of the line scans in Fig. 2 H are also shown.