FIGURE 3.

Stabilized phase separation does not affect protein partitioning. A, exemplary images of phase partitioning of a model transmembrane protein (trLAT) in untreated and 0.5 mm DCA-treated GPMVs. Red images show the localization of trLAT, which is approximately equal in both phases, relative to the fluorescent nonraft marker FAST DiO (green). B, partitioning quantified by K_p,raft (the ratio of fluorescent intensity in the raft divided by nonraft phase) was slightly, but not significantly, reduced by DCA for trLAT, a peripheral raft phase protein (GPI-anchored GFP), and an integral nonraft protein (TfR-GFP). Inset, K_p,raft of trLAT was not significantly different in control versus DCA-treated GPMVs isolated with either PFA/DTT or NEM. The higher raft phase partitioning in NEM GPMVs was expected (28). Data are mean ± S.D. (error bars) from 10–20 vesicles/condition, representative of three independent experiments.