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.
[1]
Shan Sun,et al.
Inhibition of IRF5 hyper-activation protects from lupus onset and severity.
,
2020,
The Journal of clinical investigation.
[2]
Charles A. Wartchow,et al.
Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization
,
2020,
Science Advances.
[3]
B. Barnes,et al.
Therapeutic Targeting of IRFs: Pathway-Dependence or Structure-Based?
,
2018,
Front. Immunol..
[4]
Baohong Zhang,et al.
B Cell-Intrinsic Role for IRF5 in TLR9/BCR-Induced Human B Cell Activation, Proliferation, and Plasmablast Differentiation
,
2018,
Front. Immunol..
[5]
György Vereb,et al.
Flow Cytometric FRET Analysis of Protein Interactions.
,
2018,
Methods in molecular biology.
[6]
O. Michielin,et al.
CD3 delta establishes a functional link between the T cell receptor and CD8.
,
2003,
The Journal of biological chemistry.