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. 2013 Dec 17;105(12):2733–2742. doi: 10.1016/j.bpj.2013.11.002

Figure 4.

Figure 4

FRET-detected raft affinity of PFO in DSPC/DMoPC/sterol vesicles forming coexisting Lo and Ld domains. Raft affinity of LW peptide, CT-B, and PFO in MLVs (500 μM total lipid) composed of 1:1 DSPC/DMoPC with 45 mol % cholesterol (black bars), 45 mol % 1:1 cholesterol/epicholesterol (gray bars), or 45 mol % 1:1 coprostanol/epicholesterol (striped bars) was measured in PBS pH 5.1. 2 mol % NBD-DPhPE was used as the FRET acceptor. The CLoLd/CLd ratio represents the average local acceptor concentration of acceptor around the donor (protein) in vesicles containing Lo and Ld domains (CLoLd) relative to that in a homogenous bilayer (DMoPC with 45 mol % sterol) lacking domains (CLd). CLoLd/CLd is high for a protein in Ld domains and low for a protein in Lo domains. The actual FRET (F/Fo) data are shown in Fig. S3. For vesicles containing a mixture of coprostanol and epicholesterol, in which PFO binding is not complete, corrected CLoLd/CLd values are shown (see Fig. S4 and Materials and Methods). Uncorrected F/Fo and CLoLd/CLd data are shown in Fig. S5. Average (mean) values and SD values from triplicates are shown.