Analysis of STIM expression and function in LM2-4 cells. (A) Average intracellular Ca²⁺ responses during SOCE are shown for WT (n = 137), S1KO (n = 70), S2KO (n = 73), and S1/S2 dKO (n = 87) LM2-4 cells. (B and C) Quantification of SOCE (B) and Tg-induced ER Ca²⁺ release (C) for individual cells depicted in A. The AUC was calculated from 400 to 1,200 s. (D) Analysis of SOCE in Fluo-4–loaded LM2-4 cells (n = 46) compared with MDA-MB-231 cells (n = 47). (E and F) Representative images (left) and quantification (right) of WB analysis (n = 3–4) of lysates from LM2-4 and MDA-MB-231 cells using antibodies against STIM1, STIM2, or actin, as indicated. (G) Traces of cell-wide Ca²⁺ dynamics recorded from individual WT, S1KO, S2KO, and dKO LM2-4 cells using the experimental protocol described in Fig. 5. Upper inset shows a pie chart of the fraction of cells showing global Ca²⁺ rise (blue) following UV-induced uncaging of IP3. (H) Total cell lysates were prepared from S1/S2 dKO HEK293 cells expressing either EYFP-STIM1 WT, the D76A mutant, or EGFP. WB images of the IP protein material and eluted fractions are shown, demonstrating the absence of interaction between STIM1 and IP3R2 or IP3R3. Bars show the mean ± SEM. Statistics: one-way ANOVA with Tukey’s post hoc test (B and C) or two-tailed t test (F); *P < 0.05, **P < 0.01, ***P < 0.001. AUC, area under the curve; IP, immunoprecipitated. Source data are available for this figure: SourceData FS5.