Localization of SERCA2b and IP 3 R1. (A) Immunostaining of SERCA2b and STIM1 at the whole cell level before (Control) and after store depletion with Thapsigragin. (B and D) High magnification views of the spatial organization of STIM1 and SERCA2b before (Ctr) and after store depletion (Tg) in the lateral dimension (x/y). Scale bar 500 nm. (C and E) Intensity profiles were obtained from virtual line scans in the x/y plane across ER tubules (C) and a STIM1 cluster (E). (F) Distance between the peak signals of mCh-STIM1 (center of the cluster) and the nearest SERCA2b maximum after store depletion as in panel D detected by immunofluorescence. The distance to the peak of GFP-Orai1 was used as a reference (n = 11–16; unpaired t test, P < 0.0001). (G) Pearson’s correlation coefficient (PCC) obtained before (Ctr) and after store depletion (Tg) between endogenous SERCA2b and endogenous 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, P = 0.0011). (H) Orthogonal section across the PM after store depletion illustrating the relative position and intensity of the STIM1 cluster (red) and SERCA2b (green). The arrows indicate the positions of the line analysis inside the cluster (I) and outside (O). The STIM1 and SERCA2b normalized intensities were measured over the z-axis after store depletion from inside and outside a STIM1 cluster. Scale bar 500 nm. (I) Quantification of the peak-to-peak distance in the z-axis between mCh-STIM1, Orai-GFP, and SERCA2b, inside and outside the STIM1 clusters (n = 13–16; one-way ANOVA, P < 0.0001). (J) 3D reconstruction of the relative localization of SERCA2b around the STIM1 cluster. (K) Top: Immunofluorescence for endogenous STIM1 (red) and IP3R1 (green) after store depletion with Thapsigargin. A virtual line scan (arrow) across the high-magnification image shows the separation between the SOCE cluster and “licensed” IP3R1s. Bottom: Immunofluorescence for endogenous IP3R1s and KRAP at the PM focal plane. The intensity profile measured along the white arrow in the high magnification image indicates the high degree of colocalization of the “licensed” IP3R1 and KRAP. (L) PCC summaries between STIM1, IP3R1, and KRAP (n = 8–14; one way ANOVA, P < 0.0001). (M) Histogram of the relative frequency of the nearest neighbor distance (NND) between STIM1 clusters and IP3R1 or KRAP (Outliers removed using the ROUT method and a Q value of 10%). (N) Violin plot comparing the distribution of NNDs between the center of STIM1 clusters and IP3R1 or KRAP (n = 398–803; one-way ANOVA, P < 0.0001).