FIG. 3.

baf inhibits delivery of HRV2 and fluid-phase markers to late endocytic compartments. HeLa cells preincubated without (A) or with (B) 200 nM baf (30 min, 37°C) were divided into three aliquots each. (i) Late endosomes were labeled with FITC-dextran (20 mg/ml; 3-min pulse, 12-min chase in marker-free medium); HRP (10 mg/ml) was then added for 3 min at 37°C to label the early endosomes. (ii) ³⁵S-labeled HRV2 (10⁶ cpm) was internalized for 20 min at 34°C. (iii) ³H-labeled poliovirus (6.5 × 10⁵ cpm) was bound to HeLa cells for 60 min at 4°C to label plasma membranes. The aliquots of the labeled cells were cooled and combined, washed with PBS containing 10 mM EDTA to remove plasma membrane bound HRV2, and homogenized in TEA-sucrose buffer (see Materials and Methods). Microsomes were analyzed by FFE. A total of 92 fractions were collected, and protein content, radioactivity, and HRP activity, were determined. Data are expressed as the percentage of the total amount recovered after FFE.