Summary

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em>原位</em>接近结扎分析

Published: January 16, 2017
doi:

Summary

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Abstract

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.

Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Introduction

核因子90(NF90)是一个多同种型蛋白质与许多功能,包括白细胞介素-2的后转录和miRNA生物发生1-3的调节响应于病毒感染,调节。 RBM3是一种RNA结合蛋白,参与翻译和miRNA生物发生,并且可以通过各种紧张,包括低温和缺氧4-6诱导。最近,我们发现NF90和RBM3的蛋白复合物7。 NF90和RBM3的相互作用是必不可少的调节在折叠蛋白反应7蛋白激酶RNA样内质网激酶(PERK)活性。无论NF90和RBM3主要位于细胞核中,但NF90的一小部分,并RBM3航天飞机进入细胞质并结合有彼此为特定的功能, 调节PERK活动。因此,可视化NF90-RBM3相互作用的分布在亚细胞区室,其可以指示是很重要它们在各自的舱室各种角色。

几十年前,酵母双杂交(Y2H)的开发,以检测两种蛋白质8之间的相互作用。然而,由于融合蛋白的人工建造,假阳性结果限制了这种方法的应用。很长一段时间,免疫共沉淀是分析蛋白质-蛋白质相互作用,特别是在内源性条件9的主要技术。为了分析共同免疫沉淀的蛋白质复合物,蛋白质印迹是最方便的技术,而当超灵敏性和精确性期望使用质谱法。近年来,接近连接测定已发展为一种新的方法来检测在原位 10,11在两个细胞和组织中蛋白-蛋白相互作用。

在这里,我们比较捕捉NF9菌株最流行的免疫共沉淀法和相对新颖的接近结扎测定方法在亚细胞组分0 RBM3互动。我们还讨论的优点和两种技术的局限性。

Protocol

1.免疫共沉淀种子HEK293细胞以每孔2×10 5个细胞在一6孔板的2ml Dulbecco氏改良的Eagle氏培养基(DMEM)中的补充有10%胎牛血清(FBS)和100U / mL的青霉素-链霉素(青霉素-链霉素) 。 生长细胞48小时,在37℃,5%的CO 2。 洗涤细胞用冷的磷酸盐缓冲盐水(PBS)的三倍。收获细胞,在500×g离心5分钟,离心在4℃下。 使用商业细胞核和细胞质提取试剂准备细胞核?…

Representative Results

图1表明,NF90和RBM3都是核蛋白和只有一小部分是存在于细胞质中。值得注意的是,存在用于RBM3染色阳性三个不同的频带。最小的正下方的20kDa反映RBM3的正确大小(RBM3的预测分子量为17 kDa的)。其他两个波段的起源还有待调查。与RBM3作为诱饵蛋白共免疫沉淀实验表明NF90-RBM3相互作用是主要存在于核和细胞质少数。免疫共沉淀的数据支持每个单独的蛋白质的本?…

Discussion

有几个好处,以及这两种方法的缺点。作为一个相对较新的技术中,邻近连接测定的一个明显的优点是阐明在单细胞水平,而不是一个批次异质细胞蛋白质 – 蛋白质相互作用的可行性。具有较高的幅度和分辨率( 例如 ,通过共聚焦显微镜)图像提供了通过计算单荧光斑点进行量化的可能性。相反,用Western印迹免疫共沉淀技术的常规组合只能半定量蛋白质条带,这主要是因为一个合适的负?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study was supported by the Swiss National Science Foundation (SNSF, 31003A_163305).

Materials

Dulbecco's Modified Eagle’s Medium (DMEM) Sigma D6429 High glucose
4500 mg/L
Fetal bovine serum (FBS) Gibco, Thermo Fisher Scientific 10270106
Penicillin-Streptomycin (PenStrep) BioConcept 4-01F00-H
NE-PER Nuclear and Cytoplasmic Extraction Reagents Thermo Fisher Scientific 78833
1,4-Dithiothreitol (DTT) Carl Roth 6908.3
Dynabeads Protein G Novex, Thermo Fisher Scientific 10003D
DRBP76 (NF90/NF110) antibody BD Transduction Laboratories 612154 use 1:1000 for WB and 1:100 for ICC/PLA
RBM3 antibody ProteinTech 14363-1-AP use 1:1000 for WB and 1:100 for ICC/PLA
Lamin A/C antibody Cell Signaling Technology #2032 use 1:1000 for WB 
anti-GAPDH antibody Abcam ab8245 use 1:1000 for WB 
normal rabbit IgG Santa Cruz sc-2027
anti-rabbit IgG, HRP-lined secodary antiboy Cell Signaling Technology #7074 use 1:5000 for WB 
anti-mouse HRP secondary antibody Carl Roth 4759.1 use 1:5000 for WB 
Clarity Western ECL Blotting Substrate Bio-Rad #1705060
NuPAGE Novex 4-12% Bis-Tris Gel Novex, Thermo Fisher Scientific NP0321BOX
NuPAGE LDS Sample Buffer (4x) Novex, Thermo Fisher Scientific NP0007
1,4-Dithiothreitol (DTT) CarlRoth 6908.1
NuPAGE MES SDS Running Buffer (20x) Novex, Thermo Fisher Scientific NP0002
NuPAGE Transfer Buffer (20x) Novex, Thermo Fisher Scientific NP00061
Amersham Hypond P 0.2 PVDF membrane GE Healthcare Life Sciences 10600021
Super RX X-ray film Fujifilm 4741029230
Poly-D-Lysine 8 Well Culture Slide Corning BioCoat 354632
Paraformaldehyde (PFA) Sigma P6148
Normal goat serum (NGS) Gibco, Thermo Fisher Scientific PCN5000
Goat anti-mouse IgG (H+L Antibody), Alexa Fluor 488 conjugate Thermo Fisher Scientific A-11001
Goat anti-rabbit IgG (H+L Antibody), Alexa Fluor 568 conjugate Thermo Fisher Scientific A-11011
4′, 6-Diamidin-2-phenylindol (DAPI) Sigma D9542
Duolink PLA probe Anti-mouse PLUS Sigma DUO92001
Duolink PLA  probe Anti-rabbit MINUS Sigma DUO92005
Duolink Detection Reagents Red Sigma DUO92008
Duolink Wash Buffers Fluorescence Sigma DUO82049
Duolink Mounting Medium with DAPI Sigma DUO82040
Mowiol 4-88 Sigma 81381
Microscope Olympus AX-70
CCD camera SPOT Insight 2MP Firewire
X-ray film Fujifilm Super RX
Film processing machine Fujifilm FPM-100A

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Cite This Article
Zhu, X., Zelmer, A., Wellmann, S. Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay. J. Vis. Exp. (119), e55218, doi:10.3791/55218 (2017).

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