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Fig. 4. Binding and internalization of RNase A. (A) JAR cells were incubated with BODIPY~RNase A (1 µM) for 30 minutes at 4°C, and their fluorescence was visualized directly or after a 5 minute incubation at 37°C. (B) K-562 cells were treated as in A. (C) K-562 cells were incubated with unlabeled RNase A as described in A. After a 5 minute incubation at 37°C, cells were visualized directly or fixed, and internalized RNase A was detected using an appropriate primary and secondary antibody. (D) K-562 cells were incubated with fluorescein~RNase A (open; 1 µM) or OG~RNase A (filled; 1 µM) for 20 minutes at 4°C. Fluorescence intensity was measured after a 0-6-minute incubation at 37°C with a FACScan flow cytometer. Each data point represents the mean(±s.e.) of the fluorescence from 10,000 cells in two separate experiments.