缺氧条件下培养细胞内源性核蛋白的共免疫沉淀测定
Summary
在这里, 我们描述了一个共同免疫沉淀协议, 研究在缺氧条件下内源核蛋白的蛋白质相互作用。该方法适用于低氧转录因子与转录联合调节剂相互作用的证明。
Abstract
低氧水平 (缺氧) 触发多种适应反应与缺氧诱导因子 1 (HIF-1) 复合体作为主调节器。HIF-1 由 heterodimeric 氧调节α亚基 (HIF-1α) 和组成性表达的β亚基 (HIF-1β) 也称为芳烃受体核转运体 (ARNT), 调节包括血管生成在内的不同过程中所涉及的基因、红细胞和糖酵解。HIF-1 相互作用蛋白的鉴定是了解缺氧信号通路的关键。除了调节 HIF-1α稳定性外, 缺氧还触发了许多转录因子的核易位, 包括 HIF-1α和 ARNT。值得注意的是, 目前用于研究这种蛋白质-蛋白质相互作用 (PPIs) 的方法大多是基于那些通过蛋白质过度表达人为地增加蛋白质水平的系统。蛋白质过度表达往往导致非生理结果的时间和空间文物。在这里, 我们描述了在低氧治疗后使用内源核蛋白的改进的免疫沉淀协议, 并作为概念的证明, 以显示 HIF-1α和 ARNT 之间的相互作用。在本议定书中, 缺氧细胞是在缺氧条件下收获的, Dulbecco 的磷酸盐缓冲盐水 (DPBS) 洗涤缓冲液也在使用前平衡缺氧条件, 以减轻蛋白质降解或蛋白质复合体复氧过程中的离解。此外, 核分数随后提取, 以集中和稳定内源性核蛋白, 并避免可能的假结果往往看到在蛋白质过度表达。该协议可用于证明转录因子与转录联合调节器在缺氧条件下的内源和本机相互作用。
Introduction
缺氧发生时, 没有足够的氧气供应给细胞和组织的身体。它在各种生理和病理过程中起着关键作用, 如干细胞分化、炎症和癌症1、2。缺氧诱导因子 (HIFs) 功能, heterodimers 由氧调节α亚基和组成性表达β亚基也称为 ARNT3。迄今已确定了 HIF α亚基 (HIF-1α、HIF-2α和 HIF-3α) 和三个 HIF β亚基 (ARNT/HIF-1β、ARNT2 和 ARNT3) 的三种异构体。HIF-1α和 ARNT 是无所不在表达的, 而 HIF-2α、HIF-3α、ARNT2 和 ARNT3 有更限制性的表达模式4。HIF-1 蛋白复合物是缺氧反应的关键调节因子。在缺氧条件下, HIF-1α变稳定, translocates 至细胞核, dimerizes ARNT5。随后, 这一复杂的结合到特定的核苷酸称为缺氧反应元素 (HREs), 并调节所涉及的目标基因的表达, 包括血管生成, 红细胞和糖酵解6。除了这种 “规范” 的反应, 缺氧信号通路也被称为串扰多细胞反应信号通路, 如缺口和核因子-卡伯 B (NF-nf-κb)7,8,9。
新的 HIF-1 相互作用蛋白的鉴定对于更好地理解缺氧信号通路是很重要的。与 ARNT, 这是不敏感的氧气水平和组成性表达, HIF-1α蛋白质水平是严格调节细胞氧水平。在 normoxia (21% 氧), HIF-1α蛋白质迅速退化10,11。HIF-1α在 normoxia 的短半衰期为细胞提取物中蛋白质的检测以及 HIF-1α-interacting 蛋白的鉴定提供了具体的技术挑战。此外, 一些转录因子, 包括那些 HIF-1 复合体植物常常将进入细胞核的缺氧条件下12,13,14。目前用于 PPI 研究的大多数方法都是使用非生理的蛋白质过度表达来进行的。这种蛋白过度表达通过多种机制 (包括资源超载、化学计量失衡、混杂相互作用和通路调制15、16) 引起了不同的细胞缺陷。在 PPI 研究方面, 蛋白质过度表达可能导致假阳性, 甚至是假阴性, 结果取决于蛋白质的性质和功能的抗原蛋白。因此, 目前的 PPI 研究方法必须修改, 以揭示在缺氧条件下的生理相关 PPIs。我们以前已经证明了 HIF-1 和 Ets 家族转录因子遗传结合蛋白 (GABP) 在缺氧 P19 细胞中的相互作用, 这有助于Hes1启动子对缺氧17的反应。在这里, 我们描述了一个共同免疫沉淀协议, 研究 PPIs 之间的内源性核蛋白在缺氧条件下。HIF-1α与 ARNT 的相互作用表现为概念的证明。该协议适用于证明转录因子与转录联合调节器在缺氧条件下的相互作用, 包括但不限于 HIF-1 相互作用蛋白的鉴定。
Protocol
Representative Results
Discussion
HIF-1 复合体是细胞氧稳态的主调节器, 它调节了多种细胞适应缺氧反应的基因。新的 HIF-1 相互作用蛋白的鉴定对于了解缺氧信号转导具有重要意义。免疫沉淀实验通常用于 PPIs 研究, 描绘细胞信号转导通路。然而, 蛋白质过度表达仍然被广泛使用, 这可能导致实验性工件。此外, HIF-1α是一种高度不稳定的蛋白质, 它在重氧11期间迅速降解。此外, 缺氧会触发大多数哺乳动物细胞系中 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢张庆信教授对缺氧工作站的使用。这项工作得到以下方面的支持: 新加坡教育部、教育部 1T1-02/04 和 MOE2015-T2-2-087 (Y.A.)、李港医学院、南洋理工大学开办补助金 M4230003 (P.O.B.)、瑞典研究理事会、家庭苓基金会, 诺德基金会, Stichting af Jochnick 基金会, 瑞典糖尿病协会, 氧化钪保险公司, 糖尿病研究和健康基金会, 泊位冯 Kantzow 的基础,战略研究计划在糖尿病卡罗林斯卡 Institutet, 紧急救济 ERC-2013-AdG 338936 Betalmage, 和克努特和爱丽丝拉乌尔·沃伦贝格基金会。
Materials
| Material | |||
| 1.0 M Tris-HCl Buffer, pH 7.4 | 1st BASE | 1415 | |
| Protein A/G Sepharose beads | Abcam | ab193262 | |
| Natural Mouse IgG protein | Abcam | ab198772 | |
| EDTA | Bio-Rad | 1610729 | |
| 2x Laemmli Sample Buffer | Bio-Rad | 1610737 | |
| 2-Mercaptoethanol | Bio-Rad | 1610710 | |
| Nitrocellulose Membrane | Bio-Rad | 1620112 | |
| Blotting-Grade Blocker | Bio-Rad | 1706404 | Non-fat dry milk for western blotting applications |
| 10x Tris Buffered Saline (TBS) | Bio-Rad | 1706435 | |
| 10% Tween 20 | Bio-Rad | 1610781 | |
| 10x Tris/Glycine/SDS | Bio-Rad | 1610732 | |
| 10x Tris/Glycine Buffer | Bio-Rad | 1610771 | |
| Precision Plus Protein Dual Color Standards | Bio-Rad | 1610374 | |
| Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling | 7074 | |
| Anti-mouse IgG, HRP-linked Antibody | Cell Signaling | 7076 | |
| SignalFire ECL Reagent | Cell Signaling | 6883 | |
| Dulbecco's Phosphate-Buffered Saline | Corning | 21-030-CV | |
| Phenylmethylsulfonyl fluoride (PMSF) | Merck Millipore | 52332 | |
| ARNT/HIF-1 beta Antibody | Novus Biologicals | NB100-124 | Concentration: 1.4 mg/mL |
| HIF-1 alpha Antibody | Novus Biologicals | NB100-479 | Concentration: 1.0 mg/mL |
| YY1 Antibody | Novus Biologicals | NBP1-46218 | Concentration: 0.2 mg/mL |
| Qproteome Nuclear Protein Kit | Qiagen | 37582 | Lysis buffer NL and Extraction Buffer NX1 are provied in the kit |
| GAPDH Antibody | Santa Cruz | sc-47724 | Concentration: 0.2 mg/mL |
| Glycerol (≥99%) | Sigma | G5516 | |
| Potassium chloride | Sigma | P9541 | |
| RIPA buffer | Sigma | R0278 | |
| Sodium Chloride (NaCl) | Sigma | 71376 | |
| NP-40 | Sigma | 127087-87-0 | |
| Dulbecco’s modified Eagle’s medium (DMEM, 4.5 g/L glucose) | Thermo Fisher Scientific | 11995065 | |
| Dithiothreitol (DTT) | Thermo Fisher Scientific | R0861 | |
| Fetal Bovine Serum | Thermo Fisher Scientific | 10270106 | |
| HEK293A cell line | Thermo Fisher Scientific | R70507 | |
| Methanol | Thermo Fisher Scientific | 67-56-1 | |
| Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | |
| Pierce Protease Inhibitor Tablets | Thermo Fisher Scientific | 88660 | |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | |
| QSP gel loading tip | Thermo Fisher Scientific | QSP#010-R204-Q-PK | 1-200 uL |
| Equipment/Instrument | |||
| Thick Blot Filter Paper, Precut, 7.5 x 10 cm | Bio-Rad | 1703932 | |
| Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, with Mini Trans-Blot Module and PowerPac Basic Power Supply | Bio-Rad | 1658034 | |
| 4–15% Mini-PROTEAN TGX Precast Protein Gels | Bio-Rad | 4561083 | |
| ChemiDoc XRS+ System | Bio-Rad | 1708265 | |
| I-Glove | BioSpherix | I-Glove | |
| Synergy HTX Multi-Mode Microplate Reader | BioTek | BTS1LFTA | |
| Costar 5mL Stripette Serological Pipets | Corning | 4487 | |
| Costar 10mL Stripette Serological Pipets | Corning | 4488 | |
| Costar 25mL Stripette Serological Pipets | Corning | 4251 | |
| Corning 96-Well Clear Bottom Black Polystyrene Microplates | Corning | 3631 | |
| 15mL High Clarity PP conical Centrifuge Tubes | Corning | 352095 | |
| Small Cell Scraper | Corning | 3010 | |
| Gilson Pipetman L 4-pipettes kit | Gilson | F167370 | P2, P20, P200, P1000 and accessories |
| 1.5mL Polypropylene Microcentrifuge Tubes | Greiner Bio-One | 616201 | |
| PIPETBOY acu 2 Pipettor | INTEGRA Biosciences | 155 000 | |
| Justrite Flammable Liquid Storage Cabinets | Justrite Manufacturing Co. | 896000 | |
| Vortex mixer | Labnet | S0200 | |
| CO2 incubator | NuAire | NU-5820 | |
| Orbital shakers | Stuart | SSL1 | |
| Tube rotator SB3 | Stuart | SB3 | |
| MicroCL 21R Microcentrifuge | Thermo Fisher Scientific | 75002470 | |
| Sorvall ST 16 Centrifuge | Thermo Fisher Scientific | 75004240 | |
| Tissue Culture Dishes (100 mm) | Thermo Fisher Scientific | 150350 | |
| Slide-A-Lyzer MINI Dialysis Device | Thermo Fisher Scientific | 69580 | 10K MWCO, 0.1 mL |
| Float Buoys for 0.1mL Slide-A-Lyzer MINI Dialysis Devices | Thermo Fisher Scientific | 69588 | |
| LSE Digital Dry Bath Heaters | Thermo Fisher Scientific | 1168H25 | |
| Thermo Scientific 1300 Series A2 Class II, Type A2 Bio Safety Cabinets | Thermo Fisher Scientific | 13-261-308 | |
| Software | |||
| Image Lab Software | Bio-Rad | 1709691 |
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