Localization of IP ₃ R1, STIM1, and KRAP. (A) TIRF images of STIM1, KRAP, and IP₃R1 were detected by immunofluorescence in HeLa cells after store depletion. (B) Bar chart summarizing colocalization (PCC) of IP₃R1 with KRAP but not with STIM1 (n = 16–21; unpaired t test). (C) Airyscan images or “licensed” IP₃R1–GFP and KRAP detected by immunofluorescence at the PM plane (n = 6–12; unpaired t test). (D) Relative intensities were measured along the line indicated by the white arrow in C. (E) Colocalization (PCC) between KRAP and IP₃R1–GFP before (Ctr) and after store depletion (Tg). (F) Airyscan images of IP₃R1–GFP and mCh-STIM1 inside the cell (deep ER, in control conditions) and at the PM after store depletion (CPA/Cortical). (G) Relative intensities measured along the line indicated by the white arrow in F in cells at rest. (H) Colocalization (PCC) of IP₃R1–GFP and mCh-STIM1 before and after store depletion (CPA) (n = 7; paired t test). (I) Relative intensities along the line indicated by the white arrow in F in store depleted cells. (J) Lateral distance between the mCh-STIM clusters and either IP₃R1–GFP or endogenous KRAP after store depletion (n = 6–7; unpaired t test). (K) Example of an orthogonal section through a mCh-STIM1 cluster highlighting the localization of “licensed” IP₃R1–GFP outside the cluster and “Free” IP₃R deeper within the ER away from the STIM1 cluster as illustrated by the intensity plot. The cartoon indicates the distribution of STIM1 (red) after store depletion relative to “Licensed” (L) and “Free” (F) IP₃R1–GFP. The lateral distance between STIM1 clusters and licensed IP₃R1 as measured in panel J is indicated as D. (L) Axial distance between STIM1 clusters after store depletion and KRAP, “Free” IP₃R1–GFP, or “Licensed” IP₃R1–GFP, as indicated. “Free” IP₃R1 are receptors that localized deeper in the cell and do not colocalize with KRAP.