Internalization of MPG-EGFP occurs rapidly and is inhibited at 4°C. A. EGFP or MPG-EGFP was added to HeLa cells at 40 μg/ml and cells were fixed with 4% PFA at indicated times after the acid wash. Fixed cells were visualized under an inverted fluorescence microscope. B. 40 μg/ml MPG-EGFP was added to BV2 or HeLa cells in 2-well slide chambers for 30 min, with (BV2) or without (HeLa) 5 μg/ml FM4-64. To maintain 4°C, cells and all reagents were precooled for 20 min on ice. Cells were fixed with 4% PFA for 20 min, mounted, and visualized under a confocal microscope.