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. 2009 Apr 17;284(16):10650–10658. doi: 10.1074/jbc.M809094200

FIGURE 2.

FIGURE 2.

Cdt holotoxin preferentially binds to LUVs containing cholesterol. The interaction of Cdt holotoxin with LUVs containing varying amounts of cholesterol was analyzed by FRET and SPR. Panel A shows FRET analysis of Cdt with LUVs containing varying amounts (10–40 mol %) cholesterol. Values are the mean ± S.D. (n = 3), expressed as relative % energy transfer. Results are statistically significant (p < 0.05; multivariant analysis of variance with post-hoc Scheffe test) for differences in energy transfer as cholesterol concentration is increased. Panel B shows the SPR results of Cdt interaction with LUVs. An overlay of sensorgrams shows the interaction of Cdt (10 μg/ml) with immobilized LUVs containing decreasing concentrations of cholesterol; data points were collected every 0.2 s. Data are plotted as response units versus time and are representative of three experiments. Panel C shows the results of SPR analysis for the interaction of two concentrations of Cdt (10 and 20 μg/ml) with immobilized LUVs containing 5, 10, and 20% cholesterol; data are the mean ± S.D. of three experiments and are plotted as the number of response units obtained from each sensorgram after 3 min post-injection. Results are statistically significant (p < 0.29; multivariant analysis of variance) for differences in response units as toxin concentration is increased. Panel D shows the results of SPR analysis of Cdt (20 μg/ml) with immobilized LUVs containing 20% of either cholesterol, lanosterol, ergosterol, or stigmasterol. The mean ± S.D. of the maximum response is plotted for three experiments. Results are statistically significant for differences between cholesterol and lanosterol (p < 0.001), cholesterol and ergosterol (p = 0.029), and cholesterol and stigmasterol (p = 0.029). To verify that liposomes contained comparable levels of sterol, aliquots of liposomes were extracted, and the amount of sterol was determined as described in Fig. 1; extraction yields were 7.9 μg (lanosterol), 8.6 μg (ergosterol), 7.6 μg (stigmasterol), and 7.8 μg (cholesterol).