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Fig. 8. (A) Suppression of hVps34 expression does not interfere with the endocytosis of a fluid phase marker, horseradish peroxidase (HRP). (A) Control (black) and hVps34 KD (gray) cells were incubated for the indicated periods of time with 2 mg/ml HRP in DMEM + 1% BSA. Washed cells were lysed and HRP activity was determined as described in the Materials and Methods. Each point represents the mean±s.e.m. from triplicate dishes of each cell line. (B) Suppression of hVps34 expression does not impair recycling of transferrin. Control (black) and hVps34 KD (gray) cells were allowed to internalize biotinylated transferrin for 30 minutes, then were chased with an excess of unlabeled transferrin for 15 or 30 minutes. The amounts of biotinylated transferrin in the cells and medium were determined as described in the Materials and Methods. The recycled transferrin (released into the medium) is expressed as percentage of total biotinylated transferrin. The bar graph in the inset shows the relative amount of biotinylated transferrin (normalized to calreticulin) taken up by the control (black bar) and KD (gray bar) cells after the 30 minutes pre-loading period. Each point represents the mean±s.e.m. from triplicate dishes of each cell line.
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