Fig. 3. GFP-FR and GFP-PrP both associate with DRMs, but only GFP-FR forms HMM complexes. (A) MDCK cells stably expressing GFP-FR or GFP-PrP were lysed at 4°C in buffer containing 1% Triton X-100 and separated by centrifugation until equilibrium on 5-40% sucrose-density gradients to purify Triton-X-100 insoluble microdomains. Fractions of 1 ml were collected from top (fraction 1) to bottom (fraction 12) and, after TCA-precipitation, run on SDS-PAGE and detected using anti-GFP antibody. One aliquot of each fraction was transferred onto nitrocellulose membrane, and GM1 (a typical raft-marker) was revealed by using cholera toxin conjugated to HRP. (B) Cells were lysed in buffer containing 0.4% SDS and 0.2% Triton X-100 and run through 5-30% sucrose gradients. Fractions of 500 µl were collected from the top (fraction 1) to the bottom (fraction 9) of the gradients. Proteins were TCA-precipitated and detected by western blotting using a specific GFP antibody. The position on the gradients of molecular mass markers is indicated. The graphs show the mean values of protein distribution on the gradients from three different experiments ± s.d.