Preparation of triton X-100-insoluble membrane fractions (DRMs)

The protocol was similar to that described previously (Davies et al., 2010; Kadurin et al., 2012). All steps were performed on ice. Confluent tsA-201 cells from two 175-cm (Kurshan et al., 2009) flasks were taken up in Mes-buffered saline (MBS, 25 mm Mes, pH 6.5, 150 mm NaCl, and PI) containing 1% (v/v) Triton X-100 (TX-100) (Thermo Scientific), and left on ice for 1 hr. An equal volume of 90% (w/v) sucrose in MBS was then added to a final concentration of 45% sucrose. The sample was transferred to a 13 ml ultracentrifuge tube and overlaid with 10 ml of discontinuous sucrose gradient, consisting of 35% (w/v) sucrose in MBS (5 ml) and 5% (w/v) sucrose in MBS (5 ml). The sucrose gradients were ultra-centrifuged at 140,000 ×g_avg (Beckman SW40 rotor) for 18 hr at 4°C. 1 ml fractions were subsequently harvested from the top to the bottom of the tube and aliquots of 10 μl from each fraction were analysed by SDS-PAGE and western blotting to obtain DRM profiles. When necessary, DRMs (combined peak fractions identified by the presence of flotillin-1) from the gradient were washed free of sucrose by dilution into 25 volumes of cold PBS (pH 7.4) and pelleted by ultracentrifugation at 150,000 ×g (Beckman Ti 70 rotor) for 1 hr at 4°C. TX-100-insoluble protein was resuspended in appropriate buffers as described for ³H-gabapentin binding or for deglycosylation as described above.