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Figure 1. Transferrin endocytosis and recycling are impaired by decrease of PtdIns[4,5]P2 levels. A) Single-round kinetics of transferrin uptake and recycling were determined by first incubating cells with Texas red transferrin for 30 min on ice. After washout, cells were incubated in chase medium at 37°C for various times. At each time point, cells were fixed and processed as described in Materials and Methods. Inset expands data collected within the initial 5 min. Results were normalized to total bound value and expressed as % of total bound. Data are mean ± SD; n = 5. B) Recycling of transferrin from endosomes was accessed by first loading cells with Texas red transferrin at 37°C for 60 min to saturate the entire endocytic/recycling pathway. Surface bound Texas red transferrin was removed by acid-stripping, and cells were reincubated at 37°C in chase medium for various times. In each experiment, results were normalized so that fluorescence value for Texas red transferrin at 0 min was 1.00. Inset expands data collected within the initial 10 min. Data are mean ± SD; n=5.
