American Society of Hematology

Figure 4.

$Figure 4. HSP70-macrophage interaction occurs on the lipid raft microdomain of macrophage plasma membranes. (A) Macrophages treated with exogenous HRP-labeled HSP70 (100 μg/mL, at 4°C) were washed, lysed with MBS buffer with 1% Brij98. Further fractionation using a sucrose gradient into lipid rafts (LR) (light fractions) or non-LR (heavy fractions) microdomains was undertaken. These fractions were tested by using detecting HRP activity of HRP-HSP70 complexes (as indicated); the presence of GM1 by using HRP-cholera toxin-B (as indicated); and assayed for the amount of cholesterol. (B) Similar to the conditions in panel A macrophages were treated with HSP70-HRP, and the LR-fractions were purified using Triton X-100 (as indicated). These fractions were tested for the presence of HSP70 and GM1 and were assayed for the amount of cholesterol. Further, the ability of macrophages to bind HSP70 was tested in the presence of LR-disrupting drug MCD. Macrophages were treated methyl-β-cyclodextrin (MCD; 30 mM), washed with cold PBS, and then incubated with exogenous HRP-labeled HSP70 (100 μg/mL) for 30 minutes on ice. Subsequently, macrophages were processed for purification of LRs by using Triton X-100 and tested for the presence of HSP70 and GM1 and were assayed quantitatively for the amount of cholesterol. (C) The influence of the LR-integrity on HSP70-mediated phagocytosis was tested by treating macrophages with nystatin or MCD (both drugs disrupt LRs), and a phagocytosis assay was performed. Macrophages treated with FCS served as controls to assess the effects of LR-disrupting drugs on opsonic phagocytosis. The results shown are a cumulative analysis of 3 experiments, 3 wells/group. (D) RAW264.7 macrophages were treated with HSP70 (100 μg/mL) in the presence of varying doses of MCD (as indicated). Error bars indicate one standard deviation.$

HSP70-macrophage interaction occurs on the lipid raft microdomain of macrophage plasma membranes. (A) Macrophages treated with exogenous HRP-labeled HSP70 (100 μg/mL, at 4°C) were washed, lysed with MBS buffer with 1% Brij98. Further fractionation using a sucrose gradient into lipid rafts (LR) (light fractions) or non-LR (heavy fractions) microdomains was undertaken. These fractions were tested by using detecting HRP activity of HRP-HSP70 complexes (as indicated); the presence of GM1 by using HRP-cholera toxin-B (as indicated); and assayed for the amount of cholesterol. (B) Similar to the conditions in panel A macrophages were treated with HSP70-HRP, and the LR-fractions were purified using Triton X-100 (as indicated). These fractions were tested for the presence of HSP70 and GM1 and were assayed for the amount of cholesterol. Further, the ability of macrophages to bind HSP70 was tested in the presence of LR-disrupting drug MCD. Macrophages were treated methyl-β-cyclodextrin (MCD; 30 mM), washed with cold PBS, and then incubated with exogenous HRP-labeled HSP70 (100 μg/mL) for 30 minutes on ice. Subsequently, macrophages were processed for purification of LRs by using Triton X-100 and tested for the presence of HSP70 and GM1 and were assayed quantitatively for the amount of cholesterol. (C) The influence of the LR-integrity on HSP70-mediated phagocytosis was tested by treating macrophages with nystatin or MCD (both drugs disrupt LRs), and a phagocytosis assay was performed. Macrophages treated with FCS served as controls to assess the effects of LR-disrupting drugs on opsonic phagocytosis. The results shown are a cumulative analysis of 3 experiments, 3 wells/group. (D) RAW264.7 macrophages were treated with HSP70 (100 μg/mL) in the presence of varying doses of MCD (as indicated). Error bars indicate one standard deviation.

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