Figure 8.

GIC structure is insensitive to brefeldin A. CHO cells were grown to subconfluency. Cells were then incubated in the absence (top) or presence (bottom) of 5 μM brefeldin A for 15 min at 37°C. The absence of Golgi localization of flotillin-1 and p45 was confirmed by concomitant immunofluorescence (Gkantiragas, Brügger, Stüven, Kaloyanova, Li, Löhr, Lottspeich, Wieland, and Helms, unpublished results). After incubation, the cells were directly solubilized in PEN plus 1% Triton X-100 and incubated for 30 min at 0°C. The lysate was analyzed for the presence or absence of low-density detergent insoluble complexes by isopycnic sucrose density centrifugation as described for the preparation of GICs (MATERIALS AND METHODS). The gradients were fractionated and fractions 1–9 were analyzed by SDS-PAGE and Western blotting for the presence of v-ATPase (subunit B), flotillin-1, Gαi₃, Gβ_c, caveolin-1, and p17 with the use of the corresponding antibodies.