Fig. 6. Recycling of FITC-Tfn in control and RBL-Syt III^- cells. Control RBL (a-c) or RBL-Syt III^- cells (a'-c') were grown on glass coverslips, serum starved for 1 hour and incubated with FITC-Tfn (50 µg/ml) for 1 hour at 4°C. Unbound FITC-Tfn was removed by washing with ice-cold PBS, and the cells were warmed up to 37°C to allow endocytosis. At the end of 30 minutes, cells were placed on ice and processed for immunofluorescent staining using anti Rab 11 antibodies (polyclonal, 1: 50 dilution). Cells were visualized by confocal microscopy as described under Materials and Methods. Bars, 10 µm. Data represent one of five separate experiments. (B) Tfn recycling was monitored as described in Materials and Methods. The amounts of the extra and intracellular biotin-Tfn were determined by subjecting supernatants and cell lysates to SDS-PAGE and immunoblotting. Blots were probed with HRP-conjugated streptavidin, visualized by ECL, and the intensities of the bands corresponding to biotin-Tfn were quantified by densitometry. The amount of cell-associated biotin-Tfn is presented as a percentage of total biotin-Tfn. The results presented are the average±standard deviations of five independent experiments.

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