Figure 6

Chloroquine effectively blocks the endolysosomal pathway of transduced MPG-EGFP. A. 100 μM chloroquine was added along with 40 μg/ml MPG-EGFP for 17 hr. Note the increased intensity of MPG-EGFP signal and enlarged vesicle. Confocal live images are shown at 80X. B. Vesicle fractionation was performed using HeLa cells treated with MPG-EGFP and chloroquine for 17 hr. Note that the vesicular uptake of MPG-EGFP intensified in the presence of chloroquine, and that the cytosolic fraction also contains a significant amount of MPG-EGFP. EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; Chl 50, MPG-EGFP plus 50 μM chloroquine; Chl 100, MPG-EGFP plus 100 μM chloroquine; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. The asterisk indicates that the band is a merged doublet due to the prolonged exposure, and the arrow points the expected size of MPG-EGFP. Western blotting was performed with anti-GFP antibody.