Fig. 9. FGF-2-GFP-positive microdomains are distinct from lipid rafts. CHO_FGF-2-GFP cells were grown on large culture plates for 48 hours at 37°C in the presence of doxicyclin. Following a wash procedure using PBS the cells were scraped off the culture plates in a sucrose-containing buffer. Cell breakage was achieved by using a balch homogenizer followed by differential centrifugation at 1000 g and 5000 g to sediment nuclei and cell debris. The resulting supernatant was loaded on top of a 20% (w/v) sucrose cushion and centrifuged for 60 minutes at 100,000 g in order to collect microsomal membranes freed of cytosolic proteins. The membrane sediment was resuspended in PEN buffer containing 1% (w/v) Triton X-100 at 4°C. While being resuspended several times using a 100 µl tip, the membrane suspension was kept on ice for 30 minutes. The samples were then divided and either subjected to ultracentrifugation in order to sediment detergent-insoluble complexes or adjusted to 40% (w/v) sucrose followed by flotation in a linear sucrose gradient. (A) Detergent-soluble fraction (lane 1), detergent-insoluble fraction (lane 2). (B) 14 fractions of the linear flotation gradient (lanes 1-14) with lane 1 containing the most dense sucrose fraction and lane 14 containing the lightest fraction. In the case of FGF-2-GFP and p23 detection, 60% of each fraction was TCA-precipitated and applied to the gel; in the case of caveolin-1, 15% of each fraction was TCA-precipitated and applied to the gel.