FIGURE 2.

Subcellular localization of YFP-STIM1 and Orai1. HEK-Orai1 cells transfected with YFP-STIM1 or YFP-STIM1_D76A (D76A) were fixed and imaged under basal, ER Ca²⁺ store-replete conditions or after a 10-min treatment with 1 μm Tg in 0 Ca²⁺ HBSS. A, cells were immunostained with an antibody against the ER marker, protein disulfide isomerase (PDI). Scale bars, 5 μm. B, cells were immunostained with mAb 9E10 to detect Orai1. Insets show enlarged regions indicated by boxes. Scale bars, 5 μm. C, HEK-APP cells transfected with YFP-STIM1 or YFP-STIM1_D76A were imaged using TIRF microscopy. Cells were maintained in HBSS in a humidified environment at 37 °C, and images were acquired every 15 s. After 2 min, 1 μm Tg was added in 0 Ca²⁺ HBSS to deplete Ca²⁺ stores. Representative frames from the TIRF image sequence taken before (0 min) and 10 min after the addition of Tg (12 min) are shown.