EB1 binding impedes STIM1 translocation to ER–PM junctions and Orai1 recruitment during ER Ca²⁺ depletion. (A) Translocation of YFP-STIM1 and YFP-STIM1-TRNN to ER–PM junctions after 1 µM ionomycin treatment in HeLa cells, monitored by TIRF microscopy. Bar, 2 µm. (B) Relative translocation to ER–PM junctions of YFP-STIM1 and YFP-STIM1-TRNN as described in A. 14–15 cells from three independent experiments. Mean times to half-maximal translocation (t_1/2) are indicated. (C) Relative translocation to ER–PM junctions of YFP-STIM1 subtypes and corresponding Orai1-mCherry after 1 µM TG treatment in HeLa cells, monitored by TIRF microscopy. Black, YFP-STIM1 coexpressed with Orai1-mCherry; red, STIM1-TRNN coexpressed with Orai1-mCherry. Mean traces are shown (15–23 cells from three to four independent experiments). (D) Time to the half-maximal translocation (t_1/2) of YFP-STIM1 subtypes and Orai1-mCherry as described in C. Means ± SEM are shown. **, P < 0.01; ***, P < 0.001.