Disruption of STIM1–EB1 interaction facilitated SOCE and resulted in ER Ca²⁺ store overload. (A) Relative changes in cytosolic Ca²⁺ concentration after 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Four independent experiments. (B) SOCE triggered by 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Three independent experiments. (C) Peak of SOCE in HeLa cells treated with siControl or siEB1 as described in B. Three independent experiments. (D) Slope of SOCE in HeLa cells treated with siControl or siEB1 as described in B. Three independent experiments. (E) SOCE triggered by 100 µM histamine and 1 µM TG treatment, monitored by Fura-2 ratio in HeLa cells transfected with YFP-STIM1 or YFP-STIM1-TRNN. Three independent experiments. (F) Peak of SOCE in HeLa cells transfected with YFP-STIM1 and YFP-STIM1-TRNN as described in E. Three independent experiments. (G) Slope of SOCE in HeLa cells transfected with YFP-STIM1 and YFP-STIM1-TRNN as described in E. Three independent experiments. (H) Relative changes in cytosolic Ca²⁺ concentration in response to depletion and readdition of extracellular Ca²⁺, monitored by Fura-2 ratio in HeLa cells transfected with YFP-TM, YFP-STIM1-D76A, or YFP-STIM1-D76A-TRNN. Three to four independent experiments. (I) Peak ER Ca²⁺ release by 1 µM ionomycin treatment in the absence of extracellular Ca²⁺, monitored by Fura-2 ratio in HeLa cells transfected with siControl or siEB1. Three independent experiments. (J) Relative ER Ca²⁺ levels in the resting state (phase I), after 5 µM BHQ treatment (phase II), and after BHQ washout (phase III and IV), monitored by D1ER in HeLa cells transfected with mCherry-STIM1 or mCherry-STIM1-TRNN. 16–26 cells from three independent experiments. *, P < 0.05 between STIM1 and STIM1-TRNN. (K) Relative ER Ca²⁺ levels in phase I and IV as described in J, monitored by D1ER in HeLa cells transfected with mCherry-STIM1 or mCherry-STIM1-TRNN. Means ± SEM are shown. *, P < 0.05; **, P < 0.01.