高分辨率法监测IRF3的磷酸化依赖性激活
Summary
Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.
Abstract
The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon β and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.
Introduction
在无处不在和组成性表达的转录因子，干扰素（IFN）调节因子3（IRF3）是防御病原体的第一道防线主要是通过IFNβ的诱导是关键，也可通过诱导趋化因子（CC基序）配体5（CCL5 ）和几个抗病毒蛋白，包括干扰素诱导蛋白与tetratricopeptide重复IFIT1 / 2/3 1-3。 IRF3活化已经报道以下感染与众多的病毒，或暴露于聚肌胞苷酸（聚I：C）或脂多糖（LPS）4。重要的是，研究得最多的病毒已经进化机制以逃避IRF3介导的反应，从而逃避宿主先天免疫防御5。因此，监控IRF3的激活是非常重要的，了解先天抗病毒宿主防御的分子机制，而且要确定使用病毒来抵消这种应对策略。
ENT“>许多公开的报道然而提供通过IRF3靶基因诱导的监视（IFNB1和IFIT1）进行IRF3活化和/或萤光素酶报告基因检测耦合到低分辨率十二烷基硫酸钠聚丙烯酰胺凝胶电泳的仅有限分析（SDS- PAGE）IRF3的分析，然而，大量的生化研究，各种IRF3突变体和IRF3晶体结构6-11的阐明的行为的分析有助于确定该IRF3是在经受了一组连续的翻译后修饰复合物磷酸化多个站点，设置的磷酸化参与IRF3活化似乎依赖于刺激，最有可能在细胞类型，在未感染的细胞，IRF3共存含有phosphoresidues，包括Thr135和Ser173非磷酸化和低磷酸物种，在1 -198氨基酸N-末端区域6,12-14。这种低磷酸的F积累IRF3的ORM由应力诱导剂，生长因子和DNA损伤剂6诱导。丝氨酸/苏氨酸残基在含有激活域IRF3的C末端区域的磷酸化触发由病毒以下激活，聚I：C或脂多糖以细胞类型依赖性15-17。 IRF3的C末端的磷酸化涉及组织在两个主要簇，Ser385 / Ser386和Ser396 / Ser398 / Ser402 / Thr404 / Ser405不低于7不同磷酸受体位点，每一个有助于IRF3活化通过二聚化，核积累，与CREB关联结合蛋白（CBP）/ P300共激活因子，DNA结合干扰素敏感反应元件（ISRE）共有序列和靶基因9,10,17-19的转录。 Thr390的磷酸化也被认为有助于病毒诱发IRF3激活20。 IRF3的质谱分析表明，Ser386，Thr390，Ser396和Ser402残基直接phosphorylat由κB激酶ε抑制剂（IKKε）ED / TANK结合激酶1（TBK1）激酶9,10。 也需要通过泛素化和蛋白酶体介导的降解10终止IRF3活化的磷酸化的C末端残基。这个过程也依赖于磷酸化的Ser339，这是必要的丙基异构酶的Pin1 10,11的募集。至少含有磷酸化Ser339 /三百九十六分之三百八十六残留IRF3的物种被认为是过度磷酸化形式。每个站点的确切序列和功能保持的讨论10,21的问题。这是现在很清楚，激活IRF3并不代表均匀状态，但不同的活性粒子表现出明显的磷酸化或二聚化特征存在10,22。提供IRF3活化的完整理解响应于特定的病原体，因此，有必要为characterize该活性物质种类的被诱导。诱导IRF3的靶基因，IFNB1和IFIT1，已被证明是提供了可靠的读出了IRF3激活。然而，监测这些基因的表达不IRF3的不同的激活状态之间进行区分。对IRF3的激活状态在一个特定的环境进行综合分析依赖于它的磷酸化和二聚化状态10的详细特征。磷酸化（I型），低磷酸（II型）和过度磷酸化（形式III和IV）IRF3形式6,18,23可以成功地通过减少流动性高的分辨率SDS-PAGE分析解决。单体和二聚体IRF3种可以有效地确定本地-PAGE分析。组合使用用针对不同IRF3磷酸受体位点磷酸化特异性抗体时，这些方法都大大提高。
标准协议允许的分辨率差蛋白质不允许不同IRF3磷酸化形式有效分离。在这里，我们详细描述了一种方法来实现的最高分辨率监视使用SDS-PAGE连接到本地-PAGE结合免疫使用完全和磷酸化抗体不同的病毒激活IRF3品种的诱导，在体内不同的激活之间的歧视IRF3的形式是基于它们在SDS-PAGE观察到的迁移率的变化进行的。此外，IRF3单体和二聚物可以通过非变性电泳加以区别。与免疫印迹这两个互补技术的结合被证明是一个负担得起的和敏感的方式来获取所有必要的信息IRF3的磷酸化介导的激活的全面分析。
Protocol
Representative Results
Discussion
我们在这里描述该协议包括耦合到几个使用磷酸化特异性抗体来区分IRF3的单体/二聚体和phosphoformsⅠ-Ⅳ高分辨率SDS-PAGE和天然-PAGE的组合。适当的检测这些IRF3的物种是必不可少的充分体现IRF3激活特定设置。例如，活化的巨噬细胞的LPS刺激导致形成二聚体，Ser396 / 398磷酸化IRF3的呈现一个低磷酸（II型），但并不过度磷酸（形式III和IV）中，图案在SDS-PAGE 15。使用所描述的协议，该信息可以从?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors thank previous and current members of the laboratory for development of the protocols. The work was supported by funding from the Canadian Institutes of Health Research (CIHR) [grant # MOP-130527] and from the Natural Sciences and Engineering Research Council of Canada [NSERC-355306-2012]. NG is recipient of a Tier II Canada Research Chair. AR holds a studentship from the training program of the Respiratory Health Research Network from the Fonds de la recherche du Québec-Santé (FRQS).
Materials
| F12/Ham | Life Technologies | 11765-054 | Warm in a 37°C bath before use. |
| Fetal bovine serum | Life Technologies | 12483-020 | |
| L-glutamine | Life Technologies | 25030-081 | |
| D-PBS | Life Technologies | 14190-144 | For cell culture. |
| Trypsin/EDTA 0.25 % | Life Technologies | 25200-072 | |
| Sendai virus Cantell Strain | Charles River Laboratories | 600503 | |
| Hepes | Bioshop | HEP001 | |
| Sodium chloride (NaCl) | Bioshop | SOD001.5 | |
| EDTA | Bioshop | EDT001 | |
| Glycerol | Bioshop | GLY001.1 | Cut the extreminity of the tip and pipet slowly as it is very thick. |
| IGEPAL CA-630 | Sigma-Aldrich | I7771 | Registred trademark corresponding to Octylphenoxy poly(ethyleneoxy)ethanol (Nonidet P-40) detergent |
| Leupeptin | Bioshop | LEU001 | |
| Aprotinin | Bioshop | APR600.25 | |
| Sodium fluoride | Sigma-Aldrich | 201154 | |
| Sodium orthovanadate | MP Biomedicals | 159664 | Activation of sodium orthovanadate 0.2M : 1) Ajust the pH to 10.0 using either 1 N NaOH or 1 N HCl. The starting pH of the sodium orthotovanadate solution may vary with lots of chemical. 2) The solution is yellow at pH 10.0. 3) Boil until colorless. 4) Cool to RT. 5) Reajust the pH to 10.0 and repat steps 3-4 until the solution remains colorless and stabilizes at 10.0. Store the activated sodium orthovanadate aliquots at -20°C. |
| p-nitrophenyl phosphate disodium salt hexahydrate | Sigma-Aldrich | P1585 | |
| Beta-Glycerophosphate | Sigma-Aldrich | G6376 | |
| Bio-Rad Protein Assay Reagent | Bio-Rad | 500-0006 | Cytotoxic |
| Acrylamide/Bis-Acrylamide (37.5 : 1) 40 % | Bioshop | ACR005 | Cytotoxic |
| Tris-Base | Bioshop | DEO701 | |
| Hydrochloric acid (HCl) | LabChem | LC15320-4 | Work under fume hood. Toxic and irritant. |
| Sodium dodecyl sulfate (SDS) | Bioshop | SDS001.1 | Irritant. |
| Amonium persulfate | Sigma-Aldrich | A3678 | |
| TEMED | Invitrogen | 15524-010 | Toxic and irritant. |
| Bromophenol blue | Fisher Scientific | B392-5 | |
| Beta-mercaptoethanol | Sigma-Aldrich | M6250 | Work under fume hood. Toxic to the nervous system, mucous membranes. May be toxic to upper respiratory tract, eyes, central nervous system. |
| Glycine | Bioshop | GLN001.5 | |
| Sodium deoxycholate | Sigma-Aldrich | D6750 | |
| Sodium hydroxide (NaOH) | Bioshop | SHY700 | Irritant. |
| Nitrocellulose membrane (0.45mm) | Bio-Rad | 162-0115 | |
| Acetic acid glacial | Bioshop | ACE222.4 | Work under fume hood. Toxic, irritant and flammable. |
| Red ponceau | Sigma-Aldrich | P3504 | |
| Potassium chloride (KCl) | Sigma-Aldrich | P3911 | For PBS composition for immunoblot. |
| Na2HPO4 | Bioshop | SPD307.5 | For PBS composition for immunoblot. |
| KH2PO4 | Sigma-Aldrich | P0662 | For PBS composition for immunoblot. |
| Bovine serum albumin | Sigma-Aldrich | A7906 | For PBS-T-BSA composition for immunoblot. |
| Non-fat dry milk | Carnation | ||
| Poly sorbate 20 (Tween) | MP Biomedicals | 103168 | Cut the extreminity of the tip and pipet slowly as it is very thick. |
| Anti-IRF-3-P-Ser386 | IBL-America | 18783 | Store aliquoted at -20oC. Avoid freeze/thaw. |
| Anti-IRF-3-P-Ser396 | Home made19 | Store aliquoted at -80oC. Avoid freeze/thaw. | |
| Phospho-IRF-3 (Ser396) (4D4G) | Cell Signaling Technology | 4947s | Store at -20oC. |
| Anti-IRF-3-P-Ser398 | Home made15 | Store aliquoted at -80oC. Avoid freeze/thaw. | |
| Anti-IRF-3-full length | Actif motif | 39033 | Store aliquoted at -80oC. Avoid freeze/thaw. |
| Anti-IRF3-NES | IBL-America | 18781 | Store aliquoted at -20oC. |
| Western Lightning Chemiluminescence Reagent Plus | Perkin-Elmer Life Sciences | NEL104001EA | |
| LAS4000mini CCD camera apparatus | GE healthcare | ||
| SDS-PAGE Molecular Weight Standards, Broad Range | Bio-Rad | 161-0317 | Store aliquoted at -20oC. |
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