Figures & data
Figure 1. The top panel (a) shows the chemical structures of NBD-cholesterol and NBD-PE. The lower panel (b) is a schematic representation of half of the membrane bilayer showing the localizations of the NBD groups of NBD-PE and NBD-cholesterol in phosphatidylcholine membranes. The horizontal line at the bottom indicates the center of the bilayer. Adapted and modified from reference 17.
![Figure 1. The top panel (a) shows the chemical structures of NBD-cholesterol and NBD-PE. The lower panel (b) is a schematic representation of half of the membrane bilayer showing the localizations of the NBD groups of NBD-PE and NBD-cholesterol in phosphatidylcholine membranes. The horizontal line at the bottom indicates the center of the bilayer. Adapted and modified from reference 17.](/cms/asset/1471c6eb-002e-48cd-a12b-56c10e519930/imbc_a_180266_f0001_b.gif)
Table I. Lipid contents in native hippocampal membranes as a function of increasing cholesterol depletion.
Figure 2. Effect of changing excitation wavelength on the wavelength of maximum emission of (a) NBD-cholesterol and (b) NBD-PE in native membranes (○), cholesterol-depleted (with 40 mM MβCD) membranes (▴) and liposomes of lipid extract from native membranes (□). The ratio of fluorophore to total phospholipid was maintained at 1:100 (mol/mol). The protein concentration is ∼0.04 mg/ml. See Materials and methods for other details.
![Figure 2. Effect of changing excitation wavelength on the wavelength of maximum emission of (a) NBD-cholesterol and (b) NBD-PE in native membranes (○), cholesterol-depleted (with 40 mM MβCD) membranes (▴) and liposomes of lipid extract from native membranes (□). The ratio of fluorophore to total phospholipid was maintained at 1:100 (mol/mol). The protein concentration is ∼0.04 mg/ml. See Materials and methods for other details.](/cms/asset/9bddfc8d-5ef8-4a59-a7e6-1fabffa8dfac/imbc_a_180266_f0002_b.gif)
Figure 3. (a) Effect of cholesterol depletion (by treatment with increasing concentrations of MβCD) on the magnitude of red edge excitation shift (REES) of NBD-cholesterol (○) and NBD-PE (•) in native membranes. All other conditions are as in . See Materials and methods for other details. (b) A comprehensive representation of the magnitude of red edge excitation shift (REES) obtained with NBD-cholesterol (shaded bars) and NBD-PE (white bars) in native membranes as a function of increasing cholesterol depletion (when treated with increasing concentrations of MβCD) and in liposomes of lipid extract from native membranes (data for native and cholesterol-depleted membranes taken from a). All other conditions are as in . See Materials and methods for other details.
![Figure 3. (a) Effect of cholesterol depletion (by treatment with increasing concentrations of MβCD) on the magnitude of red edge excitation shift (REES) of NBD-cholesterol (○) and NBD-PE (•) in native membranes. All other conditions are as in Figure 2. See Materials and methods for other details. (b) A comprehensive representation of the magnitude of red edge excitation shift (REES) obtained with NBD-cholesterol (shaded bars) and NBD-PE (white bars) in native membranes as a function of increasing cholesterol depletion (when treated with increasing concentrations of MβCD) and in liposomes of lipid extract from native membranes (data for native and cholesterol-depleted membranes taken from Figure 3a). All other conditions are as in Figure 2. See Materials and methods for other details.](/cms/asset/e27ee279-b7a3-48c0-8ada-2bee9c9e7b6b/imbc_a_180266_f0003_b.gif)
Figure 4. Fluorescence polarization of (a) NBD-cholesterol and (b) NBD-PE in native membranes (○), cholesterol-depleted (with 40 mM MβCD) membranes (▴) and liposomes of lipid extract from native membranes (□) as a function of excitation wavelength. The emission wavelength was 512 nm for NBD-cholesterol and 531 nm for NBD-PE. All other conditions are as in . The data points shown are the means±SE of multiple measurements. See Materials and methods for other details.
![Figure 4. Fluorescence polarization of (a) NBD-cholesterol and (b) NBD-PE in native membranes (○), cholesterol-depleted (with 40 mM MβCD) membranes (▴) and liposomes of lipid extract from native membranes (□) as a function of excitation wavelength. The emission wavelength was 512 nm for NBD-cholesterol and 531 nm for NBD-PE. All other conditions are as in Figure 2. The data points shown are the means±SE of multiple measurements. See Materials and methods for other details.](/cms/asset/95d67d10-f1b6-4444-bbd7-36925b296622/imbc_a_180266_f0004_b.gif)
Figure 5. (a) Effect of cholesterol depletion on the fluorescence polarization of NBD-cholesterol (○) and NBD-PE (•) in native membranes. Excitation was at 460 nm and emission was monitored at 512 nm for NBD-cholesterol while excitation was at 465 nm and emission was monitored at 531 nm for NBD-PE. The polarization data shown are the means±standard errors of multiple measurements. (b) A comprehensive representation of fluorescence polarization of NBD-cholesterol (shaded bars) and NBD-PE (white bars) in native membranes as a function of increasing cholesterol depletion and in liposomes of lipid extract from native membranes (data for native and cholesterol-depleted membranes taken from a). The data points shown are the means±SE of multiple measurements. All other conditions are as in . See Materials and methods for other details.
![Figure 5. (a) Effect of cholesterol depletion on the fluorescence polarization of NBD-cholesterol (○) and NBD-PE (•) in native membranes. Excitation was at 460 nm and emission was monitored at 512 nm for NBD-cholesterol while excitation was at 465 nm and emission was monitored at 531 nm for NBD-PE. The polarization data shown are the means±standard errors of multiple measurements. (b) A comprehensive representation of fluorescence polarization of NBD-cholesterol (shaded bars) and NBD-PE (white bars) in native membranes as a function of increasing cholesterol depletion and in liposomes of lipid extract from native membranes (data for native and cholesterol-depleted membranes taken from Figure 5a). The data points shown are the means±SE of multiple measurements. All other conditions are as in Figure 2. See Materials and methods for other details.](/cms/asset/341dd557-a1b3-48cd-8e64-c9f644dbfeb5/imbc_a_180266_f0005_b.gif)
Figure 6. Effect of cholesterol depletion on the mean fluorescence lifetime of NBD-cholesterol (○) and NBD-PE (•) in native membranes. Mean fluorescence lifetimes were calculated from using Equation 3. The data points shown are the means±SE of multiple measurements. All other conditions are as in . See Materials and methods for other details.
![Figure 6. Effect of cholesterol depletion on the mean fluorescence lifetime of NBD-cholesterol (○) and NBD-PE (•) in native membranes. Mean fluorescence lifetimes were calculated from Table II using Equation 3. The data points shown are the means±SE of multiple measurements. All other conditions are as in Figure 5. See Materials and methods for other details.](/cms/asset/2c5de081-5509-4e9d-b9ac-51df1ac3b734/imbc_a_180266_f0006_b.gif)
Table II. Mean fluorescence lifetimes of NBD-labeled lipids in native membranes with increasing cholesterol depletion and in lipid extractsa.