Cellular localization of VP40. (A) EGFP-VP40 robustly localizes and induces filamentous plasma membrane assembly in HEK293, A549, and HUH7.5 cells. Bars, 10 μm. (B) EGFP-VP40 cotransfected with an equimolar concentration of mCherry-Lact C2 displays colocalization signal with Lact C2 at regions of VP40 plasma membrane localization in HEK293 cells. One limitation of the colocalization analysis is local folding of the plasma membrane, which cannot be ruled out for the colocalization signals as shown. Bars, 10 μm. (C) A 5-fold difference in the mCherry-Lact C2/EGFP-VP40 transfection ratio significantly reduces VP40 plasma membrane localization in A549 cells. Bars, 10 μm. (D) EGFP-VP40 exhibits colocalization signal with the anti-phosphatidylserine antibody 4B6. CHOK-1 cells are shown. Bars, 10 μm. (E) Quantification of plasma membrane localization of VP40 plasma membrane localization in HEK293 cells (gray bars) and A549 cells (black bars) at an equimolar transfection ratio (left column) and a 5-fold increase in the Lact C2/VP40 transfection ratio. n = 3 independent experiments, with 500 cells assessed per experiment to determine SD. ***, P < 0.001.