Figure 8.

Effect of BFA on the distribution of ssHRP^{P-selectinYGVF} and ssHRP^{P-selectinKCPL} along the endocytic pathway. PC12 cells expressing either ssHRP^{P-selectinYGVF} (A–C) or ssHRP^{P-selectinKCPL} (D and E) were incubated in the presence (filled symbols) or absence (open symbols) of BFA, homogenized, and fractionated on the 1–16% Ficoll gradients. Fractions corresponding to early or late endosomes for ssHRP^{P-selectinYGVF} were collected separately and recentrifuged on 3–16% Ficoll gradients (B) or 0.9–1.85 M sucrose gradients (C), respectively. The peak of early endosomes for ssHRP^{P-selectinKCPL} was pooled (D) and recentrifuged on a 3–16% Ficoll gradient (E). In all cases, HRP activity was measured in each fraction across the gradient and divided by that in the homogenate. One representative experiment of two is shown.