American Society of Hematology

Figure 1

$Figure 1. Proteomic analysis of RBC membrane fractions shows differences in ChAc compared with healthy controls. (A) Morphology of RBCs from control and ChAc subjects. (B-E) RBCs from control (C) and ChAc were fractionated in fraction 1 (F1) corresponding to a density < 1.074, containing reticulocytes, and fraction 2 (F2) corresponding to a density > 1.092, containing acanthocytes. (B) The fractionated RBC membrane proteins were separated by 1DE and stained with colloidal Coomassie blue. The bands that were identified by mass spectrometry are indicated: β-spectrin (accession no. P11277), 48% coverage; β-spectrin (accession no. P02549), 52% coverage; band 3 (accession no. P027330), 25% coverage; band 4.1R (accession no. P11171), 22% coverage; band 4.2 (accession no. P16452), 18% coverage; β-actin (accession no. P60709), 26% coverage; GAPDH (accession no. P04406), 18% coverage; and Prx-2 (accession no. P32119), 32% coverage. The figure shows a representative of 9 experiments performed with similar results. (C) Western blot (Wb) analysis of RBC membranes separated by 1DE with specific antibodies against Prx-2, GAPDH, flotillin-1, and stomatin proteins of fractionated RBCs from controls (C) and ChAc subjects. Actin was used as loading control. The data are representative for 8 experiments. (D-E) The membrane proteins of fractionated RBCs (see “Study design”) were separated by 2DE. Twin 2DE gels were run: one stained with colloidal Coomassie and the other transferred to membrane for Western blot analysis. (D) The colloidal Coomassie blue–stained gels underwent image analysis, and differently expressed proteins were identified by mass spectrometry (see “Comparative proteomic analysis”; supplemental Methods). Red and green spots indicated the differently expressed proteins based on image analysis. The identified proteins are reported in supplemental Table 2. The figures show 1 representative experiment from a total 8 experiments performed. (E) Western blot (Wb) analysis of the 2DE maps with specific anti–protein p55 antibody on F1 RBCs from control and ChAc subjects (validating the differently expressed spots 32C, 33C, and 34C in panel 1D and supplemental Table 2), anti–flotillin-1 antibody on F1 RBCs from control and ChAc subjects (validating the differently expressed spots 40C in panel D and supplemental Table 2), and anti–protein 4.1R antibody on F2 RBCs from control and ChAc subjects (validating the differently expressed spots 64C-68C in panel D and supplemental Table 2). The figures show one representative experiment of 3 experiments performed with similar results.$

Proteomic analysis of RBC membrane fractions shows differences in ChAc compared with healthy controls. (A) Morphology of RBCs from control and ChAc subjects. (B-E) RBCs from control (C) and ChAc were fractionated in fraction 1 (F1) corresponding to a density < 1.074, containing reticulocytes, and fraction 2 (F2) corresponding to a density > 1.092, containing acanthocytes. (B) The fractionated RBC membrane proteins were separated by 1DE and stained with colloidal Coomassie blue. The bands that were identified by mass spectrometry are indicated: β-spectrin (accession no. P11277), 48% coverage; β-spectrin (accession no. P02549), 52% coverage; band 3 (accession no. P027330), 25% coverage; band 4.1R (accession no. P11171), 22% coverage; band 4.2 (accession no. P16452), 18% coverage; β-actin (accession no. P60709), 26% coverage; GAPDH (accession no. P04406), 18% coverage; and Prx-2 (accession no. P32119), 32% coverage. The figure shows a representative of 9 experiments performed with similar results. (C) Western blot (Wb) analysis of RBC membranes separated by 1DE with specific antibodies against Prx-2, GAPDH, flotillin-1, and stomatin proteins of fractionated RBCs from controls (C) and ChAc subjects. Actin was used as loading control. The data are representative for 8 experiments. (D-E) The membrane proteins of fractionated RBCs (see “Study design”) were separated by 2DE. Twin 2DE gels were run: one stained with colloidal Coomassie and the other transferred to membrane for Western blot analysis. (D) The colloidal Coomassie blue–stained gels underwent image analysis, and differently expressed proteins were identified by mass spectrometry (see “Comparative proteomic analysis”; supplemental Methods). Red and green spots indicated the differently expressed proteins based on image analysis. The identified proteins are reported in supplemental Table 2. The figures show 1 representative experiment from a total 8 experiments performed. (E) Western blot (Wb) analysis of the 2DE maps with specific anti–protein p55 antibody on F1 RBCs from control and ChAc subjects (validating the differently expressed spots 32C, 33C, and 34C in panel 1D and supplemental Table 2), anti–flotillin-1 antibody on F1 RBCs from control and ChAc subjects (validating the differently expressed spots 40C in panel D and supplemental Table 2), and anti–protein 4.1R antibody on F2 RBCs from control and ChAc subjects (validating the differently expressed spots 64C-68C in panel D and supplemental Table 2). The figures show one representative experiment of 3 experiments performed with similar results.

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