EB1 binding prevents the PB from mediating PM targeting. (A) Translocation of YFP–iMAPPER-633 to ER–PM junctions after 10 µM nocodazole (noc) treatment, monitored by confocal microscopy in HeLa cells cotransfected with EB1-mCherry. (B) Subcellular localizations of YFP–iMAPPER-633, monitored by confocal microscopy in HeLa cells transfected with siControl or siEB1. (C) EB1 protein levels detected by Western blotting using anti-EB1 antibody in HeLa cells transfected with siControl (siCtrl) or siEB1. The intensity of bands was measured by ImageJ. Relative EB1 levels are indicated. (D) YFP–iMAPPER-633–TRNN distributes to ER–PM junctions in the absence or presence of AP20187 in HeLa cells, monitored by confocal microscopy. (E) Translocation of mCherry-STIM1-TRNN to ER–PM junctions labeled by YFP–iMAPPER-633–TRNN after 1 µM TG treatment in HeLa cells, monitored by confocal microscopy. (F) Localization of YFP-STIM1 and YFP-STIM1-2K with two PB in tandem in the CT in the absence or presence of 1 µM TG in HeLa cells, monitored by TIRF microscopy. Bars, 10 µm. (G) Quantification of the puncta density of YFP-STIM1 and YFP-STIM1-2K as described in F. Means ± SEM are shown (9–13 cells from two independent experiments). (H) Basal cytosolic Ca²⁺ levels, monitored by Fura-2 ratio in HeLa cells transfected with mCherry-STIM1, mCherry-STIM1-2K, or mCherry-STIM1-D76A. Means ± SD are shown (three independent experiments). ***, P < 0.001.