Figure 3.

Increasing membrane concentrations of rab5^Q79L protects early endosome fusion from wortmannin inhibition. (a) Postnuclear supernatants containing avidin- or biotin-HRP–loaded early endosomes were separately preincubated for 10 min at 37°C with 100 μM GTP and 0, 50, 200, 500, or 1000 nM REP-1/rab5^Q79L. Correspondingly treated avidin or biotin-HRP containing fractions were then combined in a standard fusion assay and incubated at 40 min at 37°C in the presence (□) or absence (•) of wortmannin (100 nM). (b) Postnuclear supernatants were incubated as in Figure 5a. The samples were centrifuged and 13 μg of the pelleted fraction were loaded on a 13% SDS-PAGE gel. Rab5 was detected by enhanced chemiluminescence Western blotting using a rab5 monoclonal antibody. The position of BHK-21 rab5 and his₆-rab5^Q79L is shown.