HEK293 Transfection with ER-RFP Marker

HEK293 cells were purchased from Life Sciences (Carlsbad, CA) and seeded in 12-well glass-bottom plates (Cellvis, CA), cultured in DMEM/F12 media (Life Technologies, CA) supplemented with 10% Fetal Bovine Serum and 1% pen/strep. Cells were transfected as previously described^{18 (link),19 (link)}. Briefly, on the day of transfection, spent media was replaced by fresh media and HEK293 cells were transfected with plasmid constructs using ExpiFectamine 293 (Life Technologies, CA). Red Fluorescent Protein (RFP) based BacMam 2.0 constructs, specific for ER, were co-transfected the same day following manufacturer’s instructions (ThermoFisher Scientific, MA). This ER-RFP marker was used to properly identify the ER membrane, which sometimes can be easily confused for PM. To label the cell nucleus, Hoechst staining was used (Life Technologies, CA). Cells were examined with an SP8 confocal fluorescence microscope using 63 × 1.4 NA objective, Leica (Wetzlar, Germany). We captured 3 to 6 fields of view per dish, using two dishes per construct in every experiment. All experiments were performed in duplicates on different days to ensure reproducibility.