Efficient transduction of the MPG-EGFP fusion protein into various cell lines. A. 40 μg/ml MPG-EGFP was added to AN3CA or HeLa cells. NIH3T3, BV2, 293T, and HT29 were also tested (data not shown). MPG-EGFP exhibits a punctate vesicular pattern in the cytoplasm in cells. Fixation of the cells with 4% PFA (fixed) did not alter the subcellular distribution of MPG-EGFP. Cells were all observed under a confocal microscope. Fixed cells were counterstained with TO-PRO-3 iodide (shown in blue, 1:500). B. MPG-EGFP is mostly present in the endosomes. Cells were stained with 5 μg/ml FM4-64, a general marker of endocytosis (shown in red), and visualized under a confocal microscope without fixation. Overlapping of MPG-EGFP signal and FM4-64 staining generates a yellow fluorescence. C. Vesicle fractionation was performed using HeLa cells treated with EGFP or MPG-EGFP for 1 hr. -, no treatment; EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. Western blotting was performed with anti-GFP antibody.