Fig. 5. Internalization of FITC-Tfn in control and RBL-Syt III^- cells. (A) Control RBL (a,b) or RBL-Syt III^- cells (a',b') 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 time 0 (a and a') and 1.5 minutes (b and b') the cells were placed on ice and subsequently visualized by confocal microscopy as described under Materials and Methods. Bars represent 10 µm. Data represent one of three separate experiments. (B) Biotin-Tfn was allowed to internalize as described under Materials and Methods. At the end of the indicated time periods, cell-surface Tfn was removed and the amount of intracellular biotin-Tfn was determined by subjecting 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 arbitrary units. Data represent one of three separate experiments.

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