Identification of a calcitriol-regulated Sp-1 site in the promoter of human CD14 using a combined western blotting electrophoresis mobility shift assay (WEMSA).
Identification of a calcitriol-regulated Sp-1 site in the promoter of human CD14 using a combined western blotting electrophoresis mobility shift assay (WEMSA).
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DOI：
10.1251/bpo140
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发表时间：
2008-02-17
影响因子：
6.4
通讯作者：
Reiner NE
中科院分区：
文献类型：
--
作者：
Moeenrezakhanlou A;Nandan D;Reiner NE
骨化三醇（1α，25-二羟基维生素 D3）诱导单核吞噬细胞中 CD14 的表达。由于 CD14 的启动子不包含典型的维生素 D 反应元件 (VDRE)，因此其机制尚不清楚。骨化三醇已被证明可以调节转录因子 Sp-1 的活性，我们对 CD14 近端启动子的分析表明存在四个 Sp-1 样结合序列。为了确定这些位点中的哪些可能参与骨化三醇的反应，我们使用了结合电泳迁移率变动测定（EMSA）和蛋白质印迹分析（WEMSA）的系统。使用WEMSA，我们发现只有一个位于-91至-79位置（相对于转录起始位点）的类Sp-1结合序列结合了转录因子Sp1。使用对照细胞的核提取物可证明 Sp-1 与该位点的结合。值得注意的是，在用骨化三醇孵育的细胞制备的核提取物中，结合活性减弱，因此表明 Sp-1 参与骨化三醇诱导 CD14 表达。值得注意的是，这些结果表明，与 EMSA 一样，WEMSA 可以广泛应用于帮助鉴定参与调节基因表达的转录因子。然而，与传统的 EMSA 相比，WEMSA 具有许多独特的优势。用于 WEMSA 的抗体通常比 EMSA 中使用的抗体提供更明确的信号，并且这些抗体不必在天然条件下识别表位。此外，WEMSA 不需要使用标记的寡核苷酸，从而消除了与 EMSA 相关的大量费用。
Calcitriol (1α, 25-dihydroxyvitamin D3) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA. Antibodies used for WEMSA often provide less ambiguous signals than those used in EMSA, and these do not have to recognize epitopes under native conditions. In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.
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影响因子：
15.3
作者：
Hmama, Z;Nandan, D;Sly, L;Knutson, K L;Herrera-Velit, P;Reiner, N E
通讯作者：
Reiner, N E
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通讯作者：
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通讯作者：
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作者：
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