Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

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

Nuclear factor 90 (NF90) is a multi-isoform protein with numerous functions including the response to viral infection, regulation of interleukin-2 post-transcription and regulation of miRNA biogenesis ^1-3. RBM3 is an RNA-binding protein, involved in translation and miRNA biogenesis and can be induced by various stressors including hypothermia and hypoxia ^4-6. Recently, we found NF90 and RBM3 in a protein complex ⁷. The interaction of NF90 and RBM3 is essential to modulate protein kinase RNA-like endoplasmic reticulum kinase (PERK) activity in unfolded protein response ⁷. Both NF90 and RBM3 are located predominantly in the nu....

Protocol

1. Co-immunoprecipitation

1. Seed HEK293 cells at 2 x 10⁵ cells per well in one 6-well plate in 2 ml Dulbecco's Modified Eagle's Medium (DMEM) supplement with 10% fetal bovine serum (FBS) and 100 U/mL Penicillin-Streptomycin (Pen-Strep).
2. Grow cells for 48 hr at 37 °C with 5% CO₂.
3. Wash cells with cold phosphate-buffered saline (PBS) three times. Harvest cells by centrifugation at 500 x g for 5 min at 4 °C.
4. Prepare nuclear and cytoplasmic fractions using commercial nuclear and cytoplasmic extraction reagents. Follow manufacturer's instruction with some modifications.
   1. Use 3 x 1....

Results

Figure 1 demonstrates that NF90 and RBM3 are both nuclear proteins and only a small fraction is present in the cytoplasm. Notably, there are three different bands stained positive for RBM3. The smallest just below 20 kDa reflects the correct size of RBM3 (the predicted molecular weight of RBM3 is 17 kDa). The origin of the two other bands remains to be investigated. Co-immunoprecipitation experiments with RBM3 as the bait protein revealed that NF90-RBM3 interactions are p.......

Discussion

There are several benefits as well as shortcomings for both methods. As a relatively novel technique, an obvious advantage of proximity ligation assay is the feasibility to elucidate protein-protein interactions at single-cell level instead of a batch of heterogeneous cells. Images with high magnitude and resolution (e.g. by confocal microscope) provide the possibility for quantification by counting single fluorescent spots. In contrast, the conventional combination of co-immunoprecipitation technique with Weste.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle’s Medium (DMEM)	Sigma	D6429	High glucose 4,500 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 1:1,000 for WB and 1:100 for ICC/PLA
RBM3 antibody	ProteinTech	14363-1-AP	use 1:1,000 for WB and 1:100 for ICC/PLA
Lamin A/C antibody	Cell Signaling Technology	#2032	use 1:1,000 for WB
anti-GAPDH antibody	Abcam	ab8245	use 1:1,000 for WB
normal rabbit IgG	Santa Cruz	sc-2027
anti-rabbit IgG, HRP-linked secondary antibody	Cell Signaling Technology	#7074	use 1:5,000 for WB
anti-mouse HRP secondary antibody	Carl Roth	4759.1	use 1:5,000 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
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
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