RASSF4 regulates STIM1-D76A–mediated Ca²⁺ influx and its localization at ER–PM junctions. (A) HeLa cells were treated with siRASSF4 or siControl for 2 d and then were transfected with mCherry-STIM1-D76A. After 12 h, Ca²⁺ was chelated by 2 mM EGTA followed by addition of 2 mM Ca²⁺. Fura-2 ratios were determined as a function of time. Means ± SEM of >300 cells are plotted. (B) HeLa cells were transfected with mCherry-STIM1-D76A and RASSF4-YFP or a control vector. After 12 h, Ca²⁺ was chelated by 2 mM EGTA followed by addition of 2 mM Ca²⁺. Fura-2 ratios were determined as a function of time. Means ± SEM of >800 cells are plotted. (C) HeLa cells cotransfected with mCherry-STIM1-D76A and either siControl or siRASSF4 were monitored by TIRFM. Bar, 2 µm. The density of mCherry-STIM1-D76A puncta was quantified. (D) TIRF images of HeLa cells cotransfected with mCherry-STIM1-D76A and either a control vector or RASSF4-YFP. Bar, 2 µm. The density of mCherry-STIM1-D76A puncta was quantified. (C and D) Means ± SEM of 20 cells for each condition across two independent experiments are shown. (E) TIRF images of HeLa cells treated with siRASSF4 or siControl and then transfected with mCherry-STIM1-D76A. Red arrows indicate unstable STIM1-D76A puncta. Bar, 1 µm. The percentage of stable STIM1-D76A puncta was quantified. Approximately 500 puncta from six different cells for each condition across three independent experiments were analyzed. Means ± SEM are plotted. **, P < 0.01; ***, P < 0.001.