Figure 4

Erythrocytes from mice deficient in Ypel4 are ovalocytic, less deformable and have reduced Band 3 protein levels. (A) Representative scanning electron micrographs of peripheral blood (PB) from wild-type (+/+) and Ypel4-null (−/−) mice. Micrographs taken at 3000 × and 13,000 × original magnification. (B) Isolated red blood cell (RBC) long and short axes were measured using ImageJ software and RBC elongation calculated using the elongation index (EI) formula EI = (A − B)/(A + B) (n = 2). (C,D) Deformability of RBCs was measured using an Automated Rheoscope and Cell Analyzer (ARCA) applying 3 Pa (C; n = 4) or 10 Pa (D; n = 4) shear stress. The graphs show the frequency of cells (y-axis) with a specific deformability index, this index is measured as the ratio between length over width (x-axis). (E) PB analysis of Eosin-5′-Maleimide mean fluorescence intensity (MFI) in RBCs measured by flow cytometry. MFI values are normalized to mean corpuscular volume (MCV), as determined by a hematology analyzer, in each sample for accurate density assessment of Band 3 protein levels in the RBC membranes (n = 4). (F) RBCs were hemoglobin-depleted using hypotonic lysis. Membrane protein concentrations were measured and equal amounts of protein solubilized and separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). Gels were fixed in ethanol and stained with Coomassie Blue G-250 dye to visualize proteins. Representative images of one +/+ and one −/− sample is shown, first with all visible bands and then with only Bands 3, 4.1 and 4.2 visible after further separation to emphasize the visible differences in band intensities. (G) Band 3 intensities were analyzed and normalized by total protein normalization using ImageJ software. Data displayed normalized to average wild-type ratio and a paired Student’s t test was used to compare the means. Connecting lines depict age and gender matched +/+ and −/− samples (n = 4). All other data displayed as average ± SEM, *P ≤ 0.05, ** P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, a.u. arbitrary unit, kDa kilodalton.