American Society of Hematology

Figure 6.

$K3 maintains normal erythrocyte actin membrane skeleton. (A) Representative western blot analysis of major actin-binding proteins and adducin α phosphorylation in K3WT and K3KO/EpoR-cre erythrocyte ghosts. Samples from 4 different mice are shown. (B) Expression levels of actin-binding proteins in RBC ghosts from panel A, normalized to GAPDH. Densitometry of triplicate western blots was performed using Image J software. ∗P < .001; K3KO∖EpoR-cre vs K3WT mice; n = 12. (C) Densitometric analysis of phospho–adducin α (S724) normalized to total adducin α of triplicate western blots from panel A. ∗P < .001; n = 12. (D) Representative images of pellet (P) and Triton X-100–soluble (supernatant; S) fractions of erythrocyte ghosts from K3WT and K3KO/EpoR-cre mice examined using western blotting for the presence of K3, spectrins, actin, phosphorylated (S724), and total adducin α. Two mice per strain are shown. (E) Relative protein expression levels and phospho–adducin α (S724) in Triton X-100–insoluble and –soluble fractions of K3WT and K3KO/EpoR-cre erythrocytes. Optical density of each protein in Triton X-100–insoluble fraction (P) of K3WT RBC ghosts was assigned the value 1. Densitometric analysis of triplicate western blots for each protein for 3 mice per group was performed using Image J software. ∗P < .01; K3WT vs K3KO/EpoR-cre mice; n = 9. (F) Disorganized actin membrane cytoskeleton in K3KO/EpoR-cre erythrocytes. Adherent to poly-L-lysine K3WT and K3KO/EpoR-cre erythrocytes were stained for F-actin with phalloidin-Alexa Fluor 488 (green) and with anti–band 3 followed by Alexa Fluor 568 goat anti-rabbit immunoglobulin G (red). Images were taken with an HC PL APO 100×/1.47 oil objective using a Leica TCS-SP8-AOBS laser scanning confocal microscope using the LAS-X 3.5.7 software. Images are representative of 7 mice per group (scale bars, 2.5 μm). Arrows point to actin clamps and clusters.$

K3 maintains normal erythrocyte actin membrane skeleton. (A) Representative western blot analysis of major actin-binding proteins and adducin α phosphorylation in K3WT and K3KO/EpoR-cre erythrocyte ghosts. Samples from 4 different mice are shown. (B) Expression levels of actin-binding proteins in RBC ghosts from panel A, normalized to GAPDH. Densitometry of triplicate western blots was performed using Image J software. ∗P < .001; K3KO∖EpoR-cre vs K3WT mice; n = 12. (C) Densitometric analysis of phospho–adducin α (S724) normalized to total adducin α of triplicate western blots from panel A. ∗P < .001; n = 12. (D) Representative images of pellet (P) and Triton X-100–soluble (supernatant; S) fractions of erythrocyte ghosts from K3WT and K3KO/EpoR-cre mice examined using western blotting for the presence of K3, spectrins, actin, phosphorylated (S724), and total adducin α. Two mice per strain are shown. (E) Relative protein expression levels and phospho–adducin α (S724) in Triton X-100–insoluble and –soluble fractions of K3WT and K3KO/EpoR-cre erythrocytes. Optical density of each protein in Triton X-100–insoluble fraction (P) of K3WT RBC ghosts was assigned the value 1. Densitometric analysis of triplicate western blots for each protein for 3 mice per group was performed using Image J software. ∗P < .01; K3WT vs K3KO/EpoR-cre mice; n = 9. (F) Disorganized actin membrane cytoskeleton in K3KO/EpoR-cre erythrocytes. Adherent to poly-L-lysine K3WT and K3KO/EpoR-cre erythrocytes were stained for F-actin with phalloidin-Alexa Fluor 488 (green) and with anti–band 3 followed by Alexa Fluor 568 goat anti-rabbit immunoglobulin G (red). Images were taken with an HC PL APO 100×/1.47 oil objective using a Leica TCS-SP8-AOBS laser scanning confocal microscope using the LAS-X 3.5.7 software. Images are representative of 7 mice per group (scale bars, 2.5 μm). Arrows point to actin clamps and clusters.

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