Figure 7
Figure 7. NPM1 is recruited on MCP-1 promoter under LPS treatment. (A) ChIP experiment of monocyte-derived macrophages treated for indicated times with LPS using NPM1 antibody. One representative of 3 independent experiments is shown. (B) Monocyte-derived macrophages and 4-hour LPS–treated macrophage extracts were subjected to immunoprecipitation by control mouse IgG, p65 (left panel), or NPM (right panel) antibodies. The presence of p65 (top panel) or NPM (bottom panel) in the immunocomplexes was detected by immunoblotting. WB indicates Western blot. (C) Untreated macrophages and 2-hour or 4-hour LPS-treated macrophage extracts were subjected to immunoprecipitation by control mouse IgG, p65, or NPM antibodies. The presence of HDAC1 in the immunocomplexes was detected by immunoblotting. WB indicates Western blot. (D) Coexistence of p65 and NPM on MCP1 promoter was demonstrated by a sequential ChIP experiment. Chromatin was first immunoprecipitated by p65 (lane 2) or NPM (lane 3) antibody. A second round of ChIP was performed with NPM (lane 4) or IgG control (lane 5) for p65 or with p65 (lane 6) for NPM. Input is shown in lane 1. MCP1 promoter was analyzed by PCR. (E) Coexistence of NPM and p65 or HDAC1 and NPM on MCP1 promoter was demonstrated by a sequential ChIP experiment. Chromatin was first immunoprecipitated by NPM or HDAC1 antibody. A second round of ChIP was performed with p65 or IgG control for NPM or using NPM for HDAC1. MCP1 promoter was analyzed by quantitative PCR and normalized to input. (F) Primary human macrophages were transfected with the different indicated constructs. Six hours after LPS treatment, total RNA was isolated and analyzed for endogenous MCP1 mRNA expression. Absolute mRNA values were determined, normalized to L32 RNA, and reported as relative values. NT indicates nontransfected. (G) Proposed role of NPM1 in quiescent and activated macrophages. Ab differentiation into macrophages progresses, NPM1 is cleaved sequentially by caspases and cathepsin B. NPM-p30 or NPM-p20 overexpression inhibits phagocytosis and migration. On LPS activation, NPM1 proteolysis is abrogated, and NPM1 in turn inhibits cytokine expression.

NPM1 is recruited on MCP-1 promoter under LPS treatment. (A) ChIP experiment of monocyte-derived macrophages treated for indicated times with LPS using NPM1 antibody. One representative of 3 independent experiments is shown. (B) Monocyte-derived macrophages and 4-hour LPS–treated macrophage extracts were subjected to immunoprecipitation by control mouse IgG, p65 (left panel), or NPM (right panel) antibodies. The presence of p65 (top panel) or NPM (bottom panel) in the immunocomplexes was detected by immunoblotting. WB indicates Western blot. (C) Untreated macrophages and 2-hour or 4-hour LPS-treated macrophage extracts were subjected to immunoprecipitation by control mouse IgG, p65, or NPM antibodies. The presence of HDAC1 in the immunocomplexes was detected by immunoblotting. WB indicates Western blot. (D) Coexistence of p65 and NPM on MCP1 promoter was demonstrated by a sequential ChIP experiment. Chromatin was first immunoprecipitated by p65 (lane 2) or NPM (lane 3) antibody. A second round of ChIP was performed with NPM (lane 4) or IgG control (lane 5) for p65 or with p65 (lane 6) for NPM. Input is shown in lane 1. MCP1 promoter was analyzed by PCR. (E) Coexistence of NPM and p65 or HDAC1 and NPM on MCP1 promoter was demonstrated by a sequential ChIP experiment. Chromatin was first immunoprecipitated by NPM or HDAC1 antibody. A second round of ChIP was performed with p65 or IgG control for NPM or using NPM for HDAC1. MCP1 promoter was analyzed by quantitative PCR and normalized to input. (F) Primary human macrophages were transfected with the different indicated constructs. Six hours after LPS treatment, total RNA was isolated and analyzed for endogenous MCP1 mRNA expression. Absolute mRNA values were determined, normalized to L32 RNA, and reported as relative values. NT indicates nontransfected. (G) Proposed role of NPM1 in quiescent and activated macrophages. Ab differentiation into macrophages progresses, NPM1 is cleaved sequentially by caspases and cathepsin B. NPM-p30 or NPM-p20 overexpression inhibits phagocytosis and migration. On LPS activation, NPM1 proteolysis is abrogated, and NPM1 in turn inhibits cytokine expression.

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