RASSF4 regulates the localization of E-Syt2 and E-Syt3 at ER–PM junctions. (A) Localization of GFP–E-Syt2 monitored by TIRFM in siControl- or siRASSF4-treated HeLa cells. The density of GFP–E-Syt2 puncta in 20 cells across two independent experiments was quantified. Means ± SEM are plotted. (B) Localization of GFP–E-Syt3 monitored by TIRFM in siControl- or siRASSF4-treated HeLa cells. GFP–E-Syt3 puncta density in 20 cells from two independent experiments was quantified. Means ± SEM are plotted. (C) GFP–E-Syt2 at ER–PM junctions monitored by TIRFM in HeLa cells cotransfected with a control vector or RASSF4. The density of GFP–E-Syt2 puncta in 20 cells across two independent experiments was quantified. Means ± SEM are shown. (D) GFP–E-Syt3 at ER–PM junctions monitored by TIRFM in HeLa cells transfected with an empty vector or a vector for expression of RASSF4. GFP–E-Syt3 puncta density in 20 cells from two independent experiments was quantified. Means ± SEM are shown. **, P < 0.01. Bars, 2 µm.