原生聚丙烯酰胺凝胶电泳免疫溴分析内源性IRF5二聚化
Summary
介绍了一种用于分析CAL-1血浆细胞树突状细胞系内源干扰素调节因子5二聚化的西方原生体斑点方法。该协议也可以应用于其他细胞系。
Abstract
干扰素调节因子5（IRF5）是调节免疫反应的关键转录因子。它在下游激活类似收费的受体骨髓分化初级反应基因88（TLR-MyD88）信号通路。IRF5活化涉及磷酸化、二聚体化以及随后从细胞质转移到细胞核的中转位，进而诱导各种亲炎细胞因子的基因表达。IRF5活化检测检测对于研究IRF5功能及其相关途径至关重要。本文介绍了一种强大的测定，用于检测CAL-1人类血浆细胞树突状（pDC）生产线中的内源性IRF5活化。该协议包括经过修改的非脱电电泳测定，该测定可以区分IRF5的单体和二分体形式，从而提供一种经济且灵敏的方法来分析IRF5的活化。
Introduction
干扰素调节因子5（IRF5）是一种重要的转录调节器，在调节免疫反应方面起着显著作用，特别是在释放亲炎细胞因子和I型干扰素（IFN）1，2 ， 3.IRF5 的调节不当是许多自身免疫性疾病的一个促成因素，IRF5 位点中与全身性红斑狼疮、多发性硬化症、类风湿性关节炎等相关的各种多态性就可见一斑。5，6，7，8，9，10.因此，对内源性IRF5活化状态进行强有力的检测检测对于了解IRF5在生理相关细胞环境中的调节途径和下游效应至关重要。
IRF5以单核细胞、树突状细胞（DC）、B细胞和巨噬细胞1、11组成表达。与其他IRF家族转录因子一样，IRF5存在于细胞质中，处于其潜伏状态。激活后，IRF5被磷酸化并形成同构体，然后转移到细胞核中，并结合编码I型IFN和亲炎细胞因子的基因的特定调控元素，最终诱导这些基因的表达1 ，2，11，12，13.IRF5调节各种收费受体（TVR）下游的先天免疫反应，如TLR7、TLR 8和TLR 9，它们在内体中局部化，并使用MyD88发出信号1，11，14。这些TTL主要识别外来核酸物种，如单链RNA（ssRNA）和未甲基化CpGDNA，这些DNA是感染症状15、16、17、18。IRF5已被证明可以调节对细菌、病毒和真菌感染的免疫反应19，20，21。考虑到IRF5在免疫系统中的影响和多样化作用，增强或抑制IRF5活性可以成为开发治疗剂22的新途径。因此，必须制定一种协议来监测内源性IRF5的激活状态，以便彻底调查调节不同细胞类型IRF5活性的途径和机制。
据我们所知，在开发本方案之前，尚未发布用于内源性IRF5活化的生化或凝胶电泳测定。磷酸化已被证明是IRF5活化的重要的第一步，并开发了一种磷酸IRF5抗体，导致发现和确认对IRF5活性13非常重要的丝氨酸残留物。然而，虽然抗体在免疫沉淀或过度表达23时能清楚地检测磷酸化IRF5，但它未能检测出我们手中整个细胞中IRF5磷酸化（未显示数据）。二聚化是IRF5激活的下一步，迄今为止许多研究这一步的重要研究依赖于表位标记IRF5的过度表达，通常不相干的细胞类型通常不表示IRF511，12 ，24，25.先前的研究表明，二聚化IRF5不一定总是转入细胞核，因此不一定完全激活25，26。内源性IRF5核定位的测定通过成像流式细胞测定27评估IRF5的活化。这种测定已应用于对了解IRF5活性至关重要的研究中，特别是在初级或稀有细胞类型28，29中，并极大地促进了该领域的知识。然而，这种检测依赖于一个专门的仪器，这是不广泛提供给研究人员。此外，在剖析 IRF5 监管途径和识别上游调节器和路径组件时，通常需要调查激活的初始步骤。这项研究为IRF5的早期激活事件提供了一个强大而可靠的生化测定，可以在配备分子生物学工具的实验室中进行。此处描述的协议对于研究 IRF5 操作的途径和机制将非常有用，特别是与正交测定（如 IRF5 核定位的成像流细胞测定分析23） 相结合时，27，28，30.
原生聚丙烯酰胺凝胶电泳（原生PAGE）是一种广泛使用的方法，用于分析蛋白质复合物31，32。与多地硫酸钠聚丙烯酰胺凝胶电泳 （SDS-PAGE） 不同，原生 PAGE 根据蛋白质的形状、大小和电荷分离蛋白质。它也保留原生蛋白质结构，没有变性31，33，34，35。提出的协议利用了原生PAGE的这些特性，并检测了IRF5的单体和二分形式。这种方法对于检测早期激活事件尤为重要，因为没有合适的市售抗体可以检测内源磷酸化IRF5。以前，一些已发表的研究使用本机 PAGE 来评估 IRF5 二分化。然而，这些研究大多依赖于外源表位标记IRF5的过度表达来分析激活状态2，13，24，36，37.这项工作提出了一个分步协议，用于在人类血浆细胞树突状（pDC）生产线中通过改良的原生PAGE技术分析内源性IRF5二聚化，其中IRF5活性已被证明对其功能1至关重要。 38，39，40.同样的技术已经应用于其他细胞系23。
Protocol
注：此处描述的协议使用 CAL-1 pDC 细胞系，使用 Resiquimod （R848） 处理，这是 TLR7/8 的激动剂。该协议已应用于其他人类和小鼠细胞类型，包括RAW 264.7（鼠大噬细胞系）、THP-1（人类单细胞系）、BJAB（人类B细胞系）、拉莫斯（人类B细胞系）和MUTZ-3（人类树突状细胞系）23。 1. CAL-1细胞的刺激 在37°C和5%CO2无菌条件下，在含有5%胎儿牛血清（F…
Representative Results
带有抗IRF5抗体的免疫球（IB）在CAL-1细胞上进行未刺激或刺激的1μg/mL R8482小时（图1）。细胞内分物制备，并执行本机PAGE。在未刺激的CAL-1细胞中，IRF5被检测为原生PAGE上的单个带，与其单体形式相对应。在用R848治疗CAL-1细胞2小时后，IRF5单体水平随着与IRF5的二分状的缓慢迁移带的积累而同时增加而降低。 <img alt="Figure 1" class="xfigi…
Discussion
此处描述的协议是经过修改的本机 PAGE，它区分了内源性 IRF5 的单体和二分形式。很少有研究报告使用专门的成像流细胞测定技术23，27，28，30检测内源性IRF5活化。该协议使用常用技术和常用试剂和工具来评估激活早期事件的内源性 IRF5 激活状态。该协议需要对标准的本机 PAGE 协议进行简单的修改，?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了克劳彻基金会和城市大学创业基金的资助。我们感谢周实验室的所有成员对手稿的实验和批判性阅读的帮助。
Materials
| 2-Mercaptoethanol | Life Technologies, HK | 21985023 | |
| 300 W/250 V power supply 230 V AC | Life Technologies, HK | PS0301 | |
| Anti-IRF5 antibody | Bethyl Laboratories, USA | A303-385 | |
| BIOSAN Rocker Shaker (cold room safe) | EcoLife, HK | MR-12 | |
| EDTA Buffer, pH 8, 0.5 M 4 X 100 mL | Life Technologies | 15575020 | |
| Glycerol 500 mL | Life Technologies | 15514011 | |
| Glycine | Life Technologies, HK | 15527013 | |
| Goat anti-Mouse IgG DyLight 800 Conjugated Antibody | LAB-A-PORTER/Rockland, HK | 610-145-002-0.5 | |
| Goat anti-Rabbit IgG DyLight 800 Conjugated Antibody | LAB-A-PORTER/Rockland, HK | 611-145-002-0.5 | |
| Halt protease inhibitor cocktail (100x) | Thermo Fisher Scientific, HK | 78430 | |
| HEPES | Life Technologies, HK | 15630080 | |
| LI-COR Odyssey Blocking Buffer (TBS) | Gene Company, HK | 927-50000 | |
| Mini Tank blot module combo; Transfer module, accessories | Life Technologies, HK | NW2000 | |
| NativePAGE 3-12% gels, 10 well kit | Life Technologies, HK | BN1001BOX | |
| NativePAGE Running Buffer 20x | Life Technologies, HK | BN2001 | |
| NativePAGE Sample Buffer 4x | Life Technologies, HK | BN2003 | |
| NP-40 Alternative, Nonylphenyl Polyethylene Glycol | Tin Hang/Calbiochem, HK | #492016-100ML | |
| PBS 7.4 | Life Technologies, HK | 10010023 | |
| Polyvinylidene difluoride (PVDF) membrane | Bio-gene/Merck Millipore, HK | IPFL00010 | |
| Protein assay kit II (BSA) | Bio-Rad, HK | 5000002 | |
| R848 | Invivogen, HK | tlrl-r848 | |
| RPMI 1640 | Life Technologies, HK | 61870127 | |
| Sodium Chloride | ThermoFisher | BP358-1 | |
| Sodium deoxycholate ≥97% (titration) | Tin Hang/Sigma, HK | D6750-100G | |
| Tris | Life Technologies, HK | 15504020 | |
| TWEEN 20 | Tin Hang/Sigma, HK | #P9416-100ML |
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Citer Cet Article
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