Fig. 7. Detergent-insolubility of GAPR-1 is not sensitive to BFA. NRK cells were incubated for 15 minutes at 37°C in the absence or presence of BFA (5 µM). The BFA-induced redistribution of GAPR-1 into tubulo-vesicular structures was confirmed by concomitant immunofluorescence (data not shown). After incubation, the cells were directly solubilized in PEN+1% TX-100 and incubated for 30 minutes at 0°C. The lysate was analysed for the presence or absence of low-density detergent insoluble complexes by isopycnic sucrose density centrifugation as described in the Materials and Methods. The gradients were fractionated and the fractions analysed by SDS-PAGE and western blotting for the presence of GAPR-1.