Figure 1.

(A) Distributions of raft markers in sucrose density gradients. RBL-2H3 cells were lysed in buffer containing 0.05% Triton X-100, and lysates were overlaid onto discontinuous sucrose gradients. After ultracentrifugation, fractions were analyzed by SDS-PAGE and Western blotting. (B) Distribution of GM1 ganglioside and GPI-anchored Thy-1 in native RBL membranes. Cells were fixed with 2% paraformaldehyde and then Thy-1 and GM1 were labeled from the outside by using anti-Thy-1-gold (10 nm) and biotinyl-cholera toxin-avidin-gold (5 nm). Membrane sheets were prepared, postfixed with glutaraldehyde, and observed by TEM. Green outlines mark clusters of Thy-1 and red circles mark GM1. There is no significant colocalization. Bar, 100 nm. Note that 10-nm gold particles in all figures provide an internal size measurement. (C) Analysis of GM1 and Thy-1 covariance by using Ripley's K statistic. Experimental values for L(t) - t (solid red line) fall within the boundaries predicted for pairwise sets of random points (dashed lines), confirming lack of Thy-1 and GM1 colocalization.