Figure 7.

Effect of BFA on the distribution of wild-type ssHRP^P-selectin along the 1–16% Ficoll initial gradients and along the secondary gradients used for isolation of early and late endosomes. PC12 cells expressing wild-type ssHRP^P-selectin were fed with ¹²⁵I-Trn or with ¹²⁵I-EGF to monitor the position of early or late endosomes and incubated in the presence (▪) or absence (□) of BFA as described in the legend for Figure 6. After centrifugation and fractionation on 1–16% Ficoll gradients, HRP activity was determined in each fraction and divided by that in the homogenate (A). Fractions corresponding to early endosomes (5–10), as seen by distribution of ¹²⁵I-Trn (Figure 6A), were pooled together and recentrifuged on 3–16% Ficoll gradients (B). Fractions corresponding to late endosomes (13–19), as judged by distribution of ¹²⁵I-EGF internalized for 20 min at 37°C (Figure 6B), were collected and recentrifuged on 0.9–1.85 M sucrose equilibrium gradients (C). After fractionation, HRP activity was measured in each fraction and normalized to that in the homogenate.