American Society of Hematology

Figure 4.

$Figure 4. ChAc red cells showed accumulation of autophagy-related proteins, which associated with active Lyn. (A) Western blot (Wb) analysis of Ulk1 (Atg1), Atg7, Atg13, Atg4, Atg5, and LC3 in cytosolic fractions from red cells of healthy (Crtl) and ChAc subjects. Catalase was used as protein loading control. Densitometric analyses of the immunoblot bands similar to those shown are presented at right. Data are shown as means ± SD (n = 6; *P < .01 compared with healthy erythrocytes). (B-C) (Upper) Total Lyn was immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc subjects and detected with antibody to either (B) Ulk1 or (C) Atg7. (Lower) (B) Ulk1 or (C) Atg7 was immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc subjects and detected either with antibody to active Lyn (phospho-Lyn 396) or to total Lyn. The experiment shown is representative of 6 such experiments, each from an individual ChAc subject and each with similar results. IgG was used as loading control. Data are shown as means ± SD (n = 6; *P < .01 compared with healthy erythrocytes). (D) Immunoprecipitates containing equal amounts of Ulk1 (left) or Atg7 (right) were immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc subjects, and Lyn kinase activity was detected using Src-specific cdc2 peptide substrate (6-20). Data are shown as means ± SD (n = 6; *P < .01 compared with healthy erythrocytes). (E) Lyn complex from ChAc red cell cytosol was prepared as in Figure 2B, and fractions 10 to 12 were collected and resubmitted to a further centrifugation step on a glycerol gradient. Aliquots of gradient fractions underwent western blot analysis for Lyn, Ulk1, and Atg7. Arrows represent glycerol gradient molecular mass standards: glutamate dehydrogenase (62 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa). The experiment shown is representative of 8 such experiments, each from an individual ChAc subjects (see also supplemental Figure 5A for the effects of GA on high-molecular-weight complex formation).$

ChAc red cells showed accumulation of autophagy-related proteins, which associated with active Lyn. (A) Western blot (Wb) analysis of Ulk1 (Atg1), Atg7, Atg13, Atg4, Atg5, and LC3 in cytosolic fractions from red cells of healthy (Crtl) and ChAc subjects. Catalase was used as protein loading control. Densitometric analyses of the immunoblot bands similar to those shown are presented at right. Data are shown as means ± SD (n = 6; *P < .01 compared with healthy erythrocytes). (B-C) (Upper) Total Lyn was immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc subjects and detected with antibody to either (B) Ulk1 or (C) Atg7. (Lower) (B) Ulk1 or (C) Atg7 was immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc subjects and detected either with antibody to active Lyn (phospho-Lyn 396) or to total Lyn. The experiment shown is representative of 6 such experiments, each from an individual ChAc subject and each with similar results. IgG was used as loading control. Data are shown as means ± SD (n = 6; *P < .01 compared with healthy erythrocytes). (D) Immunoprecipitates containing equal amounts of Ulk1 (left) or Atg7 (right) were immunoprecipitated from red cell cytosol fraction of healthy (Ctrl, control) and ChAc subjects, and Lyn kinase activity was detected using Src-specific cdc2 peptide substrate (6-20). Data are shown as means ± SD (n = 6; *P < .01 compared with healthy erythrocytes). (E) Lyn complex from ChAc red cell cytosol was prepared as in Figure 2B, and fractions 10 to 12 were collected and resubmitted to a further centrifugation step on a glycerol gradient. Aliquots of gradient fractions underwent western blot analysis for Lyn, Ulk1, and Atg7. Arrows represent glycerol gradient molecular mass standards: glutamate dehydrogenase (62 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa). The experiment shown is representative of 8 such experiments, each from an individual ChAc subjects (see also supplemental Figure 5A for the effects of GA on high-molecular-weight complex formation).

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