American Society of Hematology

Figure 1.

$Figure 1. Chorea-acanthocytosis (ChAc) red cells show weakening of band-3 ankyrin interactions and increased cytosolic active Lyn. (A) (Upper) Total Lyn was immunoprecipitated from detergent-solubilized red cells membranes of healthy (Ctrl, control) and ChAc subjects and detected with antibody to active Lyn (phospho-Lyn 396) or total Lyn (Wb, western blot). The representative experiment shown is 1 of 16 similar experiments, each with red cells from an individual ChAc subject and each with similar results. A colloidal Coomassie-stained twin gel is shown in supplemental Figure 1. (Lower) Densitometric analysis of the immunoblots; data are shown as means ± standard deviation (SD: n = 16; *P < .05 compared with healthy red cells). (B) Band 3 was immunoprecipitated (IP) from detergent-solubilized membranes of ChAc red cells previously incubated without or with the inhibitor PP2 (10 μM). The IPs were subjected to western blot (Wb) analysis with anti-ankyrin antibody and with anti–band 3 antibody. The figure is representative of 3 independent experiments with similar results. Densitometric analysis of the immunoblot bands is shown at right. Data are shown as means ± SD (n = 6; *P < .05 compared with control untreated ChAc red cells). (C) (Upper) Total Lyn was immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc subjects and detected with antibody to active Lyn (phospho-Lyn 396) or antibody to total Lyn. The experiment shown is representative of 15 such experiments, each from an individual ChAc subject and each with similar results. Colloidal Coomassie-stained twin gel is shown in supplemental Figure 1D. (Lower) Densitometric analysis of the immunoblots; data are shown as means ± SD (n = 15; P < .05 compared with control erythrocytes). (D) (Upper) Total Lyn was immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc individuals and subjected to immunoblot detection of chorein. The experiment shown is representative of 3 such experiments, each from an individual ChAc subject and each with similar results. (Lower) Densitometric analysis of the immunoblots; data are shown as means ± SD (n = 3; *P < .05 compared with control erythrocytes). (E) Lyn kinase activity from cytosols of control or ChAc red cells was detected using Src-specific CDC2 peptide substrate (6-20) in the absence or presence of Src family-specific inhibitors dasatinib and PP2 (n = 6; *P < .05 compared with untreated red cells). (F) Red cell distribution histograms generated for red cells volume and cell hemoglobin concentration (RBC-HC) of red cells from healthy control (Ctrl) and patients with ChAc without and with PP2 treatment to inhibit Lyn activity. One experiment representative of 7 with similar results is shown. Related values for CHCM and HDW are shown in supplemental Figure 1E. (G) (Upper) Morphology of red cells from healthy and ChAc subjects with and without dasatinib (0.1 μM as in Figure 1D). Cells were imaged at 100× original magnification using a Panfluor objective with 1.30 numeric aperture on a Nikon Eclipse DS-5M camera and processed with Digital Slide (DS-L1) Nikon. One experiment representative of 7 with similar results is shown. (Lower) Quantitation of acanthocytes by brightfield microscopic analysis on ChAc red cells treated with or without dasatinib (0.1 μM). Data from 50 visual fields were collected by 2 blinded researchers. Results are presented as means ± SD (n = 6; °P < .002 compared with healthy red cells; *P < .05 compared with vehicle-treated ChAc red cells).$

Chorea-acanthocytosis (ChAc) red cells show weakening of band-3 ankyrin interactions and increased cytosolic active Lyn. (A) (Upper) Total Lyn was immunoprecipitated from detergent-solubilized red cells membranes of healthy (Ctrl, control) and ChAc subjects and detected with antibody to active Lyn (phospho-Lyn 396) or total Lyn (Wb, western blot). The representative experiment shown is 1 of 16 similar experiments, each with red cells from an individual ChAc subject and each with similar results. A colloidal Coomassie-stained twin gel is shown in supplemental Figure 1. (Lower) Densitometric analysis of the immunoblots; data are shown as means ± standard deviation (SD: n = 16; *P < .05 compared with healthy red cells). (B) Band 3 was immunoprecipitated (IP) from detergent-solubilized membranes of ChAc red cells previously incubated without or with the inhibitor PP2 (10 μM). The IPs were subjected to western blot (Wb) analysis with anti-ankyrin antibody and with anti–band 3 antibody. The figure is representative of 3 independent experiments with similar results. Densitometric analysis of the immunoblot bands is shown at right. Data are shown as means ± SD (n = 6; *P < .05 compared with control untreated ChAc red cells). (C) (Upper) Total Lyn was immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc subjects and detected with antibody to active Lyn (phospho-Lyn 396) or antibody to total Lyn. The experiment shown is representative of 15 such experiments, each from an individual ChAc subject and each with similar results. Colloidal Coomassie-stained twin gel is shown in supplemental Figure 1D. (Lower) Densitometric analysis of the immunoblots; data are shown as means ± SD (n = 15; P < .05 compared with control erythrocytes). (D) (Upper) Total Lyn was immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc individuals and subjected to immunoblot detection of chorein. The experiment shown is representative of 3 such experiments, each from an individual ChAc subject and each with similar results. (Lower) Densitometric analysis of the immunoblots; data are shown as means ± SD (n = 3; *P < .05 compared with control erythrocytes). (E) Lyn kinase activity from cytosols of control or ChAc red cells was detected using Src-specific CDC2 peptide substrate (6-20) in the absence or presence of Src family-specific inhibitors dasatinib and PP2 (n = 6; *P < .05 compared with untreated red cells). (F) Red cell distribution histograms generated for red cells volume and cell hemoglobin concentration (RBC-HC) of red cells from healthy control (Ctrl) and patients with ChAc without and with PP2 treatment to inhibit Lyn activity. One experiment representative of 7 with similar results is shown. Related values for CHCM and HDW are shown in supplemental Figure 1E. (G) (Upper) Morphology of red cells from healthy and ChAc subjects with and without dasatinib (0.1 μM as in Figure 1D). Cells were imaged at 100× original magnification using a Panfluor objective with 1.30 numeric aperture on a Nikon Eclipse DS-5M camera and processed with Digital Slide (DS-L1) Nikon. One experiment representative of 7 with similar results is shown. (Lower) Quantitation of acanthocytes by brightfield microscopic analysis on ChAc red cells treated with or without dasatinib (0.1 μM). Data from 50 visual fields were collected by 2 blinded researchers. Results are presented as means ± SD (n = 6; °P < .002 compared with healthy red cells; *P < .05 compared with vehicle-treated ChAc red cells).

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