Compartmentalization of internalized
125I-Trn and 125I-EGF in the presence or
absence of BFA. PC12 cells expressing wild-type
ssHRPP-selectin were fed with 125I-Trn for
1 h at 37°C in the presence (●) or absence (○) of 10 μg/ml
BFA added in the medium during last 30 min of incubation. Parallel
dishes were incubated with 125I-EGF for 1 h on ice in
the presence or absence of BFA for the last 10 min of incubation,
washed, and transferred to 37°C for 20 min to label late endosomes in
the presence (▴) or absence (▵) of BFA. One dish labeled with
125I-EGF for 1 h on ice was washed and allowed to
internalize ligand for 2 min at 37°C with no BFA added (A, ∗).
After removal of the noninternalized ligand, cells were rinsed with HB
and homogenized, and a PNS was centrifuged on the 1–16% Ficoll
velocity gradients (A and B). After fractionation, the peak containing
125I-Trn (fractions 5–10; A) was collected and then
recentrifuged on a 3–16% Ficoll gradient (C), whereas the peak
containing 125I-EGF (fractions 13–19; B) was collected and
recentrifuged on a 0.9–1.85 M sucrose equilibrium gradient (D) as
described in MATERIALS AND METHODS.