RASSF4 regulates STIM1 translocation to ER–PM junctions after ER Ca²⁺ depletion. (A) HeLa cells cotransfected with mCherry-STIM1 and either siControl or siRASSF4 were imaged by confocal microscopy. 1 µM TG was used for stimulation. Bar, 5 µm. (B) mCherry-STIM1 translocation to ER–PM junctions monitored by TIRFM in 1-µM TG–stimulated HeLa cells cotransfected with mCherry-STIM1 and either siControl or siRASSF4. Bar, 2 µm. The plot of mCherry-STIM1 intensity is from >12 cells for each condition across three independent experiments analyzed. Means ± SEM are plotted. **, P < 0.01. (C) HeLa cells cotransfected with mCherry-STIM1 and either RASSF4-YFP or a control vector were treated with 1 µM TG and monitored by TIRFM. Bar, 2 µm. The plot of mCherry-STIM1 intensity over time is from >12 cells for each condition across three independent experiments. Means ± SEM are plotted. *, P < 0.05.