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Research Article

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

, , , , &
Pages 195-205 | Received 26 Feb 2014, Accepted 22 Jul 2014, Published online: 15 Sep 2014

Figures & data

Figure 1. Cholesterol (A) and protein (B) content, and cholesterol/protein mass ratio (C) in DRMs obtained by treatment of erythrocytes (RBC) with TX-100, Brij 98, and Brij 58 under different conditions: 4 °C or 37 °C for intact RBC, and 4 °C for cholesterol-depleted RBC (DRMs-MβCD). Cholesterol and protein were quantified in erythrocyte membranes corresponding to 2.5 × 109 RBCs and in their DRMs obtained after centrifugation of the same amount of starting material. **p < 0.001, *p < 0.05 (unpaired Student’s t-test, n = 36).

Figure 1. Cholesterol (A) and protein (B) content, and cholesterol/protein mass ratio (C) in DRMs obtained by treatment of erythrocytes (RBC) with TX-100, Brij 98, and Brij 58 under different conditions: 4 °C or 37 °C for intact RBC, and 4 °C for cholesterol-depleted RBC (DRMs-MβCD). Cholesterol and protein were quantified in erythrocyte membranes corresponding to 2.5 × 109 RBCs and in their DRMs obtained after centrifugation of the same amount of starting material. **p < 0.001, *p < 0.05 (unpaired Student’s t-test, n = 3–6).

Figure 2. (A) Western blot of the raft markers stomatin and flotillin-2 present in DRM fractions obtained from intact (at 4 °C and 37 °C) and cholesterol-depleted (MβCD, at 4 °C) erythrocytes treated with Brij 98 and Brij 58. (B) Western blot of band 3 present in 10 fractions (from top to bottom, 1–10) obtained at 4 °C and 37 °C. Results are representative of three independent experiments.

Figure 2. (A) Western blot of the raft markers stomatin and flotillin-2 present in DRM fractions obtained from intact (at 4 °C and 37 °C) and cholesterol-depleted (MβCD, at 4 °C) erythrocytes treated with Brij 98 and Brij 58. (B) Western blot of band 3 present in 10 fractions (from top to bottom, 1–10) obtained at 4 °C and 37 °C. Results are representative of three independent experiments.

Figure 3. Distribution (mass %) of phospholipid classes (SM – sphingomyelin, PC – phosphatidylcholine, and PE – phosphatidylethanolamine) present in the samples of ghosts, TX-100 DRMs, Brij 98 DRMs, and Brij 58 DRMs analyzed by HPTLC and quantified by densitometry. DRMs were obtained from erythrocytes treated with detergents at 4 °C.

Figure 3. Distribution (mass %) of phospholipid classes (SM – sphingomyelin, PC – phosphatidylcholine, and PE – phosphatidylethanolamine) present in the samples of ghosts, TX-100 DRMs, Brij 98 DRMs, and Brij 58 DRMs analyzed by HPTLC and quantified by densitometry. DRMs were obtained from erythrocytes treated with detergents at 4 °C.

Figure 4. Distributions of fatty acids in the phosphatidylcholine (A), phosphatidylethanolamine (B), and sphingomyelin (C) fractions extracted by HPTLC from ghost membranes or DRMs, as shown in . The saturated/unsaturated fatty acids ratios in the PC, PE, and SM fractions present in ghost membranes and DRMs is given in (D). Palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), arachidonic acid (20:4), behenic acid (22:0), lignoceric acid (24:0), nervonic acid (24:1) (only detected in SM fractions). *p < 0.001 and **p < 0.05: a ghosts vs. all DRMs, b ghosts vs. TX-100 and Brij 58 DRMs, c ghosts vs. Brij 98 DRMs, d Brij 98 DRMs vs. TX-100 DRMs, e Brij 58 DRMs vs. TX-100 DRMs (unpaired Student’s t-test, n = 3). DRMs were obtained from erythrocytes treated with detergents at 4 °C.

Figure 4. Distributions of fatty acids in the phosphatidylcholine (A), phosphatidylethanolamine (B), and sphingomyelin (C) fractions extracted by HPTLC from ghost membranes or DRMs, as shown in Figure 3. The saturated/unsaturated fatty acids ratios in the PC, PE, and SM fractions present in ghost membranes and DRMs is given in (D). Palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), arachidonic acid (20:4), behenic acid (22:0), lignoceric acid (24:0), nervonic acid (24:1) (only detected in SM fractions). *p < 0.001 and **p < 0.05: a ghosts vs. all DRMs, b ghosts vs. TX-100 and Brij 58 DRMs, c ghosts vs. Brij 98 DRMs, d Brij 98 DRMs vs. TX-100 DRMs, e Brij 58 DRMs vs. TX-100 DRMs (unpaired Student’s t-test, n = 3). DRMs were obtained from erythrocytes treated with detergents at 4 °C.

Figure 5. EPR spectra of 5-SASL in intact erythrocytes (RBC), TX-100, Brij 98, and Brij 58 DRMs (A). Order parameter (S) values obtained from EPR spectra of 5- and 16-SASL (B and C, respectively) in intact (RBC) and cholesterol-depleted (MβCD) erythrocyte cells and their respective DRMs prepared at 4 °C or 37 °C with TX-100, Brij 98, and Brij 58. All EPR spectra were recorded at 25 °C. **p < 0.001, *p < 0.05 (unpaired Student’s t-test, n = 3–6).

Figure 5. EPR spectra of 5-SASL in intact erythrocytes (RBC), TX-100, Brij 98, and Brij 58 DRMs (A). Order parameter (S) values obtained from EPR spectra of 5- and 16-SASL (B and C, respectively) in intact (RBC) and cholesterol-depleted (MβCD) erythrocyte cells and their respective DRMs prepared at 4 °C or 37 °C with TX-100, Brij 98, and Brij 58. All EPR spectra were recorded at 25 °C. **p < 0.001, *p < 0.05 (unpaired Student’s t-test, n = 3–6).

Figure 6. Sequence of images of erythro-GUVs in the presence of (A) 0.1 mM Brij 98 and (B) 0.5 mM TX-100. The time shown in each snapshot is relative to the moment when the erythro-GUVs were added to the detergent suspension. At least 20 GUVs were followed in the presence of Brij 98 and 100 GUVs in the presence of TX-100. The scale bars represent 10 μm.

Figure 6. Sequence of images of erythro-GUVs in the presence of (A) 0.1 mM Brij 98 and (B) 0.5 mM TX-100. The time shown in each snapshot is relative to the moment when the erythro-GUVs were added to the detergent suspension. At least 20 GUVs were followed in the presence of Brij 98 and 100 GUVs in the presence of TX-100. The scale bars represent 10 μm.
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