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Fig. 4. Transferrin recycling is inhibited in cells expressing GFPRab11b mutants. (A,B) Binding (on ice) of Alexa-568-transferrin to control and to cells transiently expressing GFPRab11b-GDP. (C,D) Transferrin retained in the GFPRab11b-GDP-expressing cell after a 30 minute chase at 37°C. The outlines of individual cells in the island are indicated by dotted lines. (A,C) GFPRab11b-GDP-expressing cells identified by GFP fluorescence; (B,D) Alexa-568-transferrin. (E) An assay for specificity of transferrin binding and uptake by PtK1 cells. Cells were incubated with increasing concentrations (10, 50, 100 and 250 µg/ml each) of fluorescently labeled Alexa568-transferrin and FITC-dextran either at 37°C or on ice. Each bar represents average fluorescence intensity of approximately 100 cells per condition. Each condition was assayed in three different wells, four fields of view per well and analyzed using Thora software. (F) Transferrin recycling in cells transiently expressing GFPRab11b-GDP, measured as fluorescent intensity of Alexa-568-transferrin retained inside the cells at the end of a 30-minute chase period, and expressed as a percentage of total transferrin bound at the beginning of experiment. (G) Recycling efficiency in cells transiently expressing GFPRab11b-GDP, expressed as a percentage of neighboring control cells values. All data are mean ± s.e.m., calculated from at least three experiments, 20-40 cells per experiment. (H) Correlation between the GFPRab11b-GDP expression level in a stable GFPRab11b-GDP cell line and transferrin retained inside the cells after a 1 hour recycling period. Data for 40 cells from a representative experiment are shown.
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