American Society of Hematology

Figure 2.

$Figure 2. In ChAc red cells, increased active Lyn is present in cytosol fractions coelutes with HSP90/HSP70 chaperone components and requires active heat shock protein HSPs. (A) Western blot (Wb) analysis of HSP70, HSP90, and Cdc37 in cytosolic fractions from red cells of healthy (Crtl) and ChAc subjects. Catalase served as protein loading control. Densitometric analysis (arbitrary units) of the immunoblot bands similar to those shown are presented at right: the data are shown as means ± SD (n = 5; *P < .01 compared with healthy erythrocytes). (B) Cytosol from control or ChAc red cells was loaded onto a linear glycerol gradient (10-40%) and centrifuged 18 hours at 100 000g in an SW60Ti rotor (Beckman Coulter) at 4°C. Eighteen fractions (200 μL each) were collected from above and analyzed by immunoblotting with antibodies to Lyn, HSP90, HSP70, and Cdc37. Arrows mark the glycerol gradient molecular mass standards: glutamate dehydrogenase (62 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa). The experiment shown is representative of 6 such experiments, each from an individual healthy or ChAc subject. Densitometric analysis is reported in supplemental Figure 2A. Data on HSP70 are shown as supplemental Figure 2B. (C) Healthy (Ctrl, control) or ChAc red cells were treated for 30 minutes at 4°C in the absence or presence of 1 μM geldanamycin (GA) or 0.1 μM GST/SH3-Lyn, and cytosol was further subjected to immunoprecipitation by anti-HSP90 and anti-Lyn antibodies and assayed for Lyn and HSP90, respectively, by western blot analysis (see also supplemental Figure 2B for HSP70).$

In ChAc red cells, increased active Lyn is present in cytosol fractions coelutes with HSP90/HSP70 chaperone components and requires active heat shock protein HSPs. (A) Western blot (Wb) analysis of HSP70, HSP90, and Cdc37 in cytosolic fractions from red cells of healthy (Crtl) and ChAc subjects. Catalase served as protein loading control. Densitometric analysis (arbitrary units) of the immunoblot bands similar to those shown are presented at right: the data are shown as means ± SD (n = 5; *P < .01 compared with healthy erythrocytes). (B) Cytosol from control or ChAc red cells was loaded onto a linear glycerol gradient (10-40%) and centrifuged 18 hours at 100 000g in an SW60Ti rotor (Beckman Coulter) at 4°C. Eighteen fractions (200 μL each) were collected from above and analyzed by immunoblotting with antibodies to Lyn, HSP90, HSP70, and Cdc37. Arrows mark the 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 6 such experiments, each from an individual healthy or ChAc subject. Densitometric analysis is reported in supplemental Figure 2A. Data on HSP70 are shown as supplemental Figure 2B. (C) Healthy (Ctrl, control) or ChAc red cells were treated for 30 minutes at 4°C in the absence or presence of 1 μM geldanamycin (GA) or 0.1 μM GST/SH3-Lyn, and cytosol was further subjected to immunoprecipitation by anti-HSP90 and anti-Lyn antibodies and assayed for Lyn and HSP90, respectively, by western blot analysis (see also supplemental Figure 2B for HSP70).

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