Chromatin Immunoprecipitation Analysis of Bortezomib-Mediated Inhibition of NFκB Recruitment to IL-1β and TNFα Gene Promoters in Human Macrophages
Chromatin Immunoprecipitation Analysis of Bortezomib-Mediated Inhibition of NFκB Recruitment to IL-1β and TNFα Gene Promoters in Human Macrophages
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DOI：
10.1007/978-1-4939-0928-5_29
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发表时间：
2014-01-01
期刊：
影响因子：
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通讯作者：
Vancurova, Ivana
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文献类型：
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作者：
Sanacora, Shannon;Chang, Tzu-Pei;Vancurova, Ivana
白细胞介素-1 β（IL-1）和肿瘤坏死因子-α（TNF）是重要的促炎细胞因子，参与免疫应答、炎症、组织修复和肿瘤进展的介导。IL-1和TNF表达的调节在转录水平上由转录因子NF κ B介导。蛋白酶体抑制剂硼替佐米（BZ）抑制NF κ B B活性已被用作多发性骨髓瘤和其他血液恶性肿瘤的一线治疗。在本章中，我们描述了一个协议，使用染色质免疫沉淀（ChIP）来分析NF κ B B招聘内源性IL-1和TNF启动子在BZ处理的人巨噬细胞。与BZ抑制IL-1和TNF的mRNA水平相对应，我们表明BZ抑制p65 NF κ B募集到IL-1和TNF启动子。本研究专门使用U937巨噬细胞，但该方案可以很容易地修改，以分析其他细胞类型中NF κ B募集的调节。
Interleukin-1 beta (IL-1) and tumor necrosis factor-alpha (TNF) are important pro-inflammatory cytokines involved in the mediation of the immune response, inflammation, tissue repair, and tumor progression. Regulation of IL-1 and TNF expression is mediated at the level of transcription by the transcription factor NF kappa B. Inhibition of NF kappa B activity by the proteasome inhibitor bortezomib (BZ) has been used as a frontline therapy in multiple myeloma and other hematological malignancies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the NF kappa B recruitment to endogenous IL-1 and TNF promoters in BZ-treated human macrophages. Corresponding to the BZ-suppressed mRNA levels of IL-1 and TNF, we show that BZ inhibits p65 NF kappa B recruitment to IL-1 and TNF promoters. This study specifically uses U937 macrophages, but the protocol could be easily modified to analyze the regulation of NF kappa B recruitment in other cell types.