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. 2021 May 20;11(10):e4021.
doi: 10.21769/BioProtoc.4021.

Intracellular IRF5 Dimerization Assay

Cherrie D Sherman  1 , Betsy J Barnes  1
Affiliations

Intracellular IRF5 Dimerization Assay

Cherrie D Sherman et al. Bio Protoc. .

Abstract

The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5 dimers, THP-1 cells are incubated in the presence or absence of the IRF5-targeted CPP (IRF5-CPP) inhibitor for 30 min then the cells are stimulated with R848 for 1 h. Cell lysates are separated by native-polyacrylamide gel electrophoresis (PAGE) and IRF5 dimers are detected by immunoblotting with IRF5 antibodies. To detect endogenous interactions between IRF5 and FITC-labeled IRF5-CPP, an in-cell fluorescence resonance energy transfer (FRET) assay is used. In this assay, THP-1 cells are left untreated or treated with FITC-IRF5-CPP conjugated inhibitors for 1 h. Next, cells are fixed, permeabilized, and stained with anti-IRF5 and TRITC-conjugated secondary antibodies. Transfer of fluorescence can be measured and calculated as FRET units. These methods provide rapid and accurate assays to detect IRF5 molecular interactions.

Keywords: Dimerization; FRET; IRF5; IRF5-CPP; Molecular interaction; Native-PAGE; Polyacrylamide gel electrophoresis.

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Conflict of interest statement

Competing interestsDisclosures: J.A.D., S.-L.T., and D.S. are inventors on patent application US20160009772A1 assigned to F. Hoffmann–La Roche AG. Application status abandoned as of 12 May 2019 as a matter of public record. Financial disclosures related to companies: G.C., C.-C.S., J.Q., M.D., and J.A.D. are employees of EMD Serono Research and Development Institute Inc. S.H. is employee of BMS. F.M. is the author of patent “Cell penetrating peptides & methods of identifying cell penetrating peptides” (WO2014001229A2) filed by F. Hoffmann–La Roche. J.A.D., N.F., A.F.H., K.-S.H., F.M., D.S., and S.-L.T. are authors of patent “Cell penetrating peptides which bind IRF5” (US20160009772A1) filed by Hoffmann–La Roche Inc.

Figures

Figure 1.
Figure 1.. Overview of native-PAGE method designed for detecting endogenous IRF5 homodimers
Figure 2.
Figure 2.. Schematic diagram of native-PAGE set up
Figure 3.
Figure 3.. Native-PAGE.
On lane 1 control – untreated THP-1, lane 2 treated with 1 μM R848, lane 3 treated with 1 μM IRF5-CPP2 and 1 μM R848, lane 4 treated with 10 μM IRF5-CPP2 and 1 μM R848, lane 5 treated with 1 μM IRF5-CPP5 and 1 μM R848, and lane 6 treated with 10 μM IRF5-CPP5 and 1 μM R848.
Figure 4.
Figure 4.. Binding of IRF5-CPPs to IRF5.
THP-1 cells were incubated with FITC-IRF5-CPP2, FITC-IRF5-CPP5, FITC-IRF5-CPP8, or FITC-IRF5-CPP9 for 1 h, followed by permeabilization and staining for intracellular IRF5 with anti-IRF5 and tetramethyl rhodamine isothiocyanate (TRITC) antibodies. FRET units were calculated from fluorescence emissions.
See this image and copyright information in PMC

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