Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Abstract

Citation: Verhelst, J., De Vlieger, D., Saelens, X. Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein. J. Vis. Exp. (98), e52871, doi:10.3791/52871 (2015). AbstractStudying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP-Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described coimmunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually. Video LinkThe video component of this article can be found at

Functional Comparison of Mx1 from Two Different Mouse Species Reveals the Involvement of Loop L4 in the Antiviral Activity against Influenza A Viruses

“…We previously showed that A2G Mx1 inhibits the interaction between NP and PB2 and that this inhibition correlates with the antiviral activity (18,35). Therefore, we investigated the ability of both Mx1 proteins to suppress the interaction between PB2 and NP.…”

Equine Mx1 Restricts Influenza A Virus Replication by Targeting at Distinct Site of its Nucleoprotein

“…The HEK293T cell line was selected as the first choice for the transfection of plasmids throughout the study except for virus infection experiments because the HEK293T cells are well used in previous studies [6,38,49,50]. Since influenza viruses are the pathogens that cause major respiratory tract infections, the lung epithelial cells might be used for in vitro analysis of host-virus interaction at the cellular level.…”

“…HEK293T cells were then co-transfected with p3XFLAG-tagged eqMx1 (4 µg) together with PB1, PB2 (2 µg each), PA, FF-Luc (1 µg each), and NP expression plasmids either from (1) H7N9, and its mutants H7N9-S34G and N52H; or (2) H3N8 JL89 and its mutants JL89-G34S and H52N; or (3) an empty control vector (4 µg each) by using PolyJet™ In Vitro DNA transfection reagent (SignaGen, Rockville, MD, USA) according to the manufacturer's instructions. At24 hr post-transfection, co-immunoprecipitation was performed as previously described [38]. Briefly, transfected cells were lysed using an ice-cold lysis buffer (50 mM of Tris-HCL [pH 8], 150 mM of NaCl, 5 Mm of EDTA, and 1% NP-40), protease inhibitors (1:100), and freshly prepared 2 M N-ethylmaleimide (NEM) at 1:80 (Sigma Aldrich, St. Louis, MO, USA) and centrifuged at 4 • C for 10 min at 13,000× g. Post centrifugation, using Anti-FLAG M2 magnetic beads (Sigma Aldrich, St. Louis, MO, USA, M8823) or Anti-NP magnetic beads (made by incubation of MCE protein A/G magnetic beads along with the NP antibody from our lab) the lysates were co-incubated for 2 hr at 4 • C. Using a magnetic separator, the resins were harvested and washed thrice with cold PBS.…”

CAFs‐derived MFAP5 promotes bladder cancer malignant behavior through NOTCH2/HEY1 signaling

“…The soluble fraction (the cell lysate) was transferred to a fresh 1.5 mL microcentrifuge tube and kept on ice. Co‐IP was performed immediately according to the protocol by Judith Verhelst 40 . Anti‐MFAP5, anti‐DLL3, and anti‐NOTCH2 antibody was used for Co‐IP.…”