蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位
Summary
细胞间连接是乳腺阶段特异性功能和发育的必需条件。该手稿提供了一个详细的方法，用于研究蛋白质 – 蛋白质相互作用（PPI）和使用鼠乳腺的共定位。这些技术允许调查不同发育阶段的细胞间连接之间的物理联系的动力学。
Abstract
细胞间的相互作用在保持组织完整性和乳腺不同隔室之间的屏障方面发挥关键作用。这些相互作用由在相邻细胞之间形成核酸的连接蛋白提供。功能蛋白质错位和与其结合配偶体的物理缔合减少可导致功能丧失，从而导致器官功能障碍。因此，在正常和疾病相关组织中鉴定蛋白质定位和蛋白质 – 蛋白质相互作用（PPI）对于找到导致疾病发展或发育状态改变的新证据和机制是至关重要的。该手稿提供了两步法评估鼠乳腺中的PPI。在方案1中，描述了使用针对感兴趣的蛋白质产生的抗体，随后用荧光染料标记的二抗进行共免疫荧光（co-IF）的方法。虽然共同所有为了证明蛋白质的接近程度，它确实有可能研究其物理相互作用。因此，在协议第2节中提供了共免疫沉淀（co-IP）的详细方案。该方法用于确定蛋白质之间的物理相互作用，而不确认这些相互作用是直接还是间接的。在过去几年中，共同中心和共同IP技术已经证明，细胞间连接的某些组成部分共同定位并相互作用，从而在乳腺发育过程中产生不同阶段依赖的连接性核酸。
Introduction
乳腺生长发育主要发生于出生后。这种器官在整个哺乳动物的生殖生活中不断地改造自己1 。成年乳腺上皮由腔内上皮细胞的内层和由基底膜2包围的主要由肌上皮细胞组成的基底细胞外层组成。为了对乳腺结构和发育的良好综述，读者可以参考Sternlicht 1 。通过间隙（GJ），紧密（TJ）和粘附（AJ）结的细胞 – 细胞相互作用对于腺体1,3,4,5,6的正常发育和功能是必需的。这些小鼠乳腺结节的主要成分是Cx26，Cx30，Cx32和Cx43（GJ）;紧密连接-1，-3，-4和-7和ZO-1（TJ）;和E-钙粘蛋白，P-钙粘蛋白和β-连环蛋白（AJ） 7,8 。这些不同连接蛋白的表达水平在乳腺发育过程中以阶段依赖的方式变化，这表明差异细胞 – 细胞相互作用的要求9 。 GJ，TJ和AJ在结构和功能上相互连接并将其他结构或调节蛋白连接到相邻细胞的相邻位点，从而产生连接性连接10 。连接关系的组成可以影响与下面的细胞骨架的桥接，以及连接通透性和稳定性，因此可以影响腺体8,9,10,11的功能。细胞间连接的组成部分存在于连接核酸中，或者彼此相互作用最近使用共免疫荧光（co-IF）和共免疫沉淀（co-IP） 9分析了乳腺发育的不同发育阶段。虽然其他技术允许评估蛋白质之间的功能关联，但这些方法在本手稿中没有提出。
由于蛋白质仅仅单独起作用，研究蛋白质 – 蛋白质相互作用（PPI），如信号转导和生物化学级联，对许多研究人员来说是必不可少的，可以提供关于蛋白质功能的重要信息。 Co-IF和微观分析有助于评估一些共享相同亚细胞空间的蛋白质。然而，目标的数量受到必须在不同动物中提高的抗体以及通过使用配备有不同波长激光器的共聚焦显微镜和用于复用的光谱检测器的抗体的限制。 Co-IP确认或揭示高亲和力的物理相互作用een两个或更多的蛋白质位于蛋白质复合物内。尽管开发了诸如荧光共振能量转移（FRET） 12和邻近连接测定（PLA） 13等新技术 ，其可以同时检测蛋白质的定位和相互作用，但共有IP仍然是一种适当和可负担的技术来研究内源蛋白。
本手册中描述的逐步方法将有助于蛋白质定位和PPI的研究，并指出在研究乳腺中的内源PPI时应避免的缺陷。该方法从对每种技术所需组织的不同保存程序的介绍开始。第1部分介绍了如何在三个步骤中研究蛋白质共定位：i）乳腺切片，ii）使用co-IF技术对不同蛋白质的双重或三重标记，以及iii）成像蛋白质定位。第2部分显示如何沉淀内源性蛋白质并以三个步骤鉴定其相互作用的蛋白质：i）裂解物制备，ii）间接蛋白质免疫沉淀，以及iii）通过SDS-PAGE和Western印迹结合配偶体鉴定。该方案的每一步都针对啮齿动物乳腺组织进行了优化，并产生了高质量，特异性和可重复的结果。该方案也可用作其他组织或细胞系中PPI研究的起点。
Protocol
本研究中使用的所有动物方案均由大学动物护理委员会（INRS-Institut Armand-Frappier，Laval，Canada）批准。 识别蛋白质共定位 从组织到显微镜片 注意：组织和部分应在干冰上处理。 从动物上清除乳腺（有关此程序的完整描述，请参阅Plante 等人 ）。 将切除的组织嵌入冷冻/安装介质中的干冰上。添加足够的培养基覆盖腺体。当培养基固?…
Representative Results
为了确定GJ，AJ和TJ组分是否可以在乳腺中相互作用，首先进行共IF测定。分别用特异性抗体探测Cx26，GJ蛋白和β-Catenin，一种AJ蛋白，分别用荧光团缀合的小鼠-647（绿色，假色）和山羊-568（红色）抗体（ 图1B和C ） 。数据显示，在哺乳期第7天（L7），它们在小鼠乳腺的上皮细胞的细胞膜上共同定位，如黄色所反映的（ 图1D ）。其次，?…
Discussion
通过连接的细胞 – 细胞相互作用是许多器官（如乳腺）的正常功能和发育所必需的。研究表明，连接蛋白可以调节彼此的功能和稳定性，并通过在细胞膜10处彼此束缚来激活信号转导。目前手稿中提出的方案提供了有关连接蛋白差异表达，定位和正常鼠腺发育相互作用的有趣发现9 。鉴于连接蛋白定位对于蛋白质的功能至关重要，并且由于已知其与支架蛋白和许…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
知识产权由加拿大自然科学与工程研究理事会资助（NSERC＃418233-2012）资助;魁北克省魁北克省圣诞老人（FRQS），魁北克省乳腺癌基金会职业生涯奖，以及加拿大创新基金会授予的领导者资助。 ED获得了Fondation大学生Armand-Frappier奖学金。
Materials
| Mice strain and stage | St. Constant, Quebec, Canada | C57BL/6 Femals; pregnancy day 18 (P18) and lactation day 14 (L14), Charles River Canada | |
| PBS 10X (stock) | 1) Dissolve 80g NaCl (F.W.: 58.44), 2g KCl (F.W. 74.55), 26.8g Na2HPO4•7H2O (F.W. 268.07) and 2.4g KH2PO4 (F.W.:136.09) in 800ml distilled water. 2) Adjust the PH to 7.4 3) Add water to reach to the 1 litre final volume. |
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| TBS 10X (stock) | 1) Dissolve 60.5g TRIS, 87.6g NaCl in 800ml distilled water. 2) Adjust the PH to 7.5 3) Add water to reach to the 1 litre final volume. |
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| Name | Company | Catalog Number | Comments |
| Part 1-Immunofluorescence | |||
| Freezing media | VWR International, Ville Mont-Royal, QC, Canada | 95067-840 | VMR frozen sections compound |
| Microtome | Mississauga, ON, Canada | 956640 | Microm HM525, Thermo fisher scientific HM525 NX Cryostat 115V 60Hz |
| Blades | C.L. Sturkey, Inc. Les Produits Scientifiques ESBE St-Laurent, QC, Canada | BLM1001C | High profile gold coated blades |
| Pap pen | Cedarlane, Burlington, ON, Canada | 8899 | Super PAP Pen, Thermo fisher scientific |
| Microscopic slides | Fisher Scientific, Burlington, ON, Canada | 12-550-15 | Fisherbrand Superfrost Plus Microscope Slides |
| Formaldehyde | BioShop Canada Inc, Burlington, ON, Canada | FOR201.1 | Forlmadehyde |
| Bovine Serum Albumin (BSA) | Santa Cruz Biotechnology, Inc, California, USA | ||
| Blocking solution | 3% BSA in TBS | ||
| Wash solution | TBS-Tween 20 0.1% | ||
| Polysorbate 20 | Oakville, ON, Canada | P 9416 | Tween 20, Sigma-Aldrich |
| Mounting media | Cedarlane, Burlington, ON | 17984-25(EM) | Fluoromount-G |
| First & secondary antibodies | Cell Signaling, Beverly, MA, USA | Mentioned in Column D | E-Cadherin (4A2) Mouse mAb (#14472s) 1/50 (Cell Signaling) with anti-mouse IgG Fab2 Alexa Fluor 555 (#4409s), Cell Signaling |
| First & secondary antibodies | Life technologies, Waltham, MA, USA & Cell Signaling, Beverly, MA, USA | Mentioned in Column D | Claudin-7 (#34-9100) 1/100 (Life Technologies) with anti-rabbit IgG Fab2 Alexa Fluor 488 (#4412s) (Cell Signaling) |
| First & secondary antibodies | Santa Cruz Biotechnology, Inc, California, USA; Fischer Scientific, Burlington, ON, Canada | Mentioned in Column D | β-Catenin Antibody (C-18): sc-1496 (SANTA CRUZ) with anti-Goat IgG (H+L) Alexa Fluor 568 (#A11057), Molecular Probe (Fisher Scientific) |
| First & secondary antibodies | Life technologies, Waltham, MA, USA & Cell Signaling, Beverly, MA, USA | Mentioned in Column D | Connexin26 (#33-5800) 1/75 (Life Technologies) with anti-mouse IgG Fab2 Alexa Fluor 647 (#4410s) |
| Nuclei stain | Fisher Scientific, Burlington, ON, Canada | D1306 | DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) 1/1000 in PBS |
| Fluorescent microscope | Nikon Canada, Mississauga, On, Canada | Nikon A1R+ confocal microscopic laser equipped with a spectral detector | |
| Software of IF images analysis | Nikon Canada, Mississauga, On, Canada | NIS-elements software (version 4) | |
| Name | Company | Catalog Number | Comments |
| Part 2-Immunoprecipitation | |||
| Triple-detergent Lysis buffer (100ml) pH=8.0 | 1) Mix 50mM TRIS (F.W. :121.14), 150mM NaCl (F.W. :58.44), 0.02% Sodium Azide, 0.1% SDS, 1% NONIdET P40, 0.5% Sodium Deoxycholate in 80ml distilled H2O. 2) Adjust the PH to 8.0 with HCl 6N (~0.5ml). 3) Adjust the volume to 100ml. Keep it in fridge. At the day of protein extraction, use 1/100 NaVo3, 1/100 protease/phosphatase inhibitor and 1/25 NAF in calculated amount of Triple detergent lysis buffer: Sodium Fluorid (stock) solution 1.25M (F.W. 41.98), Sodium Orthovanadate (stock) Solution 1M (F.W.: 183.9) |
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| Protease/phosphatase inhibitor | Fisher Scientific, Burlington, ON | 78441 | Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) |
| Protein dosage | Thermo Scientific, Rockford, Illinois, USA | 23225 | Pierce BCA protein assay kit |
| Tissue grinder | Fisher Scientific, Burlington, ON | FTH-115 | Power 125, Model FTH-115 |
| Magnetic beads and stand | Millipore, Etobicoke, ON, Canada | PureProteome Protein G Magnetic Bead System (LSKMAGG02) | |
| Wash solution for IP | PBS or PBS-Tween20 0.1% depending to the step | ||
| Primary antibodies for immunoprecipitation | Cell Signaling, Beverly, MA, USA | Mentioned in coulmn D | IgG Rabbit (rabbit (DA1E) mAb IgG Isotype control (#3900s) (Cell Signaling) 0.5 µl/200 µl |
| Primary antibodies for immunoprecipitation | Cell Signaling, Beverly, MA, USA | Mentioned in coulmn D | IgG Mouse mouse (G3A1) mAb IgG Isotype control (#5415s) (Cell Signaling) 0.5 µl/200 µl |
| Primary antibodies for immunoprecipitation | Sigma-Aldrich, Oakville, ON, Canada | Mentioned in coulmn D | Connexin43 (#C6219) (Sigma-Aldrich) 4 µl/200 µl |
| Primary antibodies for immunoprecipitation | Cell Signaling, Beverly, MA, USA | Mentioned in coulmn D | E-cadherin (4A2) Mouse mAb (#14472s) (Cell Signaling) 1 µl/200 µl |
| Laemmli buffer | BIO-RAD, Mississauga, Ontario, Canada | 1610747 | 4x Laemmli Sample Buffer (Add β-mercaptoethanol following manufacturer recommendation) |
| Acidic glycine | Fisher Scientific, Burlington, ON | PB381-5 | 0.2 M glycine; adjust pH=2.5 with HCl |
| Tris | Fisher Scientific, Burlington, ON | BP152-1 | 1 M (pH=8) |
| SDS-PAGE acrylamide gels | BIO-RAD, Mississauga, ON, Canada | 1610180 -5 | TGX Stain-Free FastCast Acrylamide Solutionss (7.8%, 10%, 12%) |
| Running buffer 10x | BIO-RAD, Mississauga, ON, Canada | 1704272 | Tris 30.3g/glycine 144.1g /SDS 10g in 1 litre distilled water |
| Membranes | BIO-RAD, Mississauga, ON, Canada | 1704272 | PVDF membranes, Trans-Blot Turbo RTA Mini PVDF Transfer Kit |
| Transfer method | BIO-RAD, Mississauga, ON, Canada | 1704155 | Trans-Blot Turbo Transfer System |
| Dry Milk | Smucker Food of Canada Co, Markham, ON, Canada | Fat Free Instant Skim Milk Powder, Carnation | |
| Blocking solution for blots | 5% dry milk in TBS-Tween 20 0.1% | ||
| Washing solutions for blots | TBS-Tween 20 0.1% | ||
| Primary and secondary antibodies for blots (10ml) | Sigma-Aldrich, Oakville, Ontario & Abcam, Toronto, ON, Canada | Mentioned in column D | Connexin43 (#C6219) (Sigma-Aldrich) 1/2500 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada) |
| Primary and secondary antibodies for blots (10ml) | Cell Signaling, Beverly, MA, USA & Abcam, Toronto, ON, Canada | Mentioned in column D | E-cadherin (24E10) rabbit mAb 1/1000 (#3195s) (Cell Signaling) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada) |
| Primary and secondary antibodies for blots (10ml) | Life technologies, Waltham, MA, USA & Abcam, Toronto, ON, Canada | Mentioned in column D | Claudin-7 (#34-9100) (Life technologies) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada) |
| Primary and secondary antibodies for blots (10ml) | Life technologies, Waltham, MA, USA & Abcam, Toronto, ON, Canada | Mentioned in column D | Claudin3 (#34-1700) (Life technologies) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada) |
| Luminol solution for signal detection on blots | BIO-RAD, Mississauga, ON, Canada | 1705061 | Clarity Western ECL Blotting Substrate |
| Imaging blots | BIO-RAD, Mississauga, ON, Canada | 1708280 | ChemiDoc MP imaging system |
| Analayzing blots | BIO-RAD, Mississauga, ON, Canada | ImageLab 5.2 software | |
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Citazione di questo articolo
Dianati, E., Plante, I. Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands. J. Vis. Exp. (123), e55772, doi:10.3791/55772 (2017).