Background

Interferon regulatory factor 5 (IRF5) is a transcription factor that regulates pathogen-induced innate and acquired immune responses downstream of Toll-like receptor (TLR), retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and B cell receptor (BCR) (De et al., 2017; Thompson et al., 2018; Banga et al., 2020). IRF5 has been implicated in the pathogenesis of systemic lupus erythematosus due to its role in regulating the expression of proinflammatory cytokines such as IFN-α, IL-6, TNF-α, and IL-12 and pathogenic autoantibody production (Song et al., 2020). In an unstimulated condition, IRF5 is generally localized in the cytoplasm as a monomer (Thompson et al., 2018). Activation of the above receptors triggers cellular signaling cascades. IRF5 undergoes post-translational modification, which eventually leads to homodimerization, a critical event prior to nuclear translocation (Thompson et al., 2018). Here, we describe two methods designed for detecting IRF5 molecular interactions.

A native-PAGE method is used to detect endogenous IRF5 homodimers. THP-1 cells are incubated with or without (1 and 10 μM) IRF5-CPP inhibitors for 30 min, and subsequently stimulated with 1 μM of R848 (a TLR7 ligand) or left unstimulated for 1 h. Next, cell lysates are run on native polyacrylamide gel electrophoresis (PAGE) and IRF5 dimerization is detected by immunoblotting with IRF5 and HRP-conjugated secondary antibodies. The intracellular fluorescence resonance energy transfer (FRET) assay is used to detect binding of endogenous IRF5 to FITC-IRF5-CPP conjugated inhibitors through a FRET signal (Banga et al., 2020; Song et al., 2020). FRET is a technique designed for detecting molecular interaction in which the excited molecule (the donor) transfers non-radiative energy to another molecule (the acceptor) within a distance of ~1-10 nm (Doucey et al., 2003; Ujlaky-Nagy et al., 2018; Banga et al., 2020). THP-1 cells are incubated with or without (1 and 10 μM) IRF5-CPP inhibitors for 1 h. The untreated and FITC-IRF5-CPP- treated THP-1 cells are then fixed, permeabilized, and stained with anti-IRF5, anti-IRF3, or anti-IRF7 (other IRF family members) antibodies and TRITC secondary antibodies. Cell-associated fluorescence is measured on a BioTek Synergy Neo2 at 525 nm upon excitation at 488 nm (E1), at 600 nm upon excitation at 540 nm (E2), and at 600 nm upon excitation at 488 nm (E3). The transfer of fluorescence is calculated as FRET units as follows: FRET unit = (E3_both − E3_none) − ([E3_TRITC − E3_none) × (E2_both/E2_TRITC]) − ([E3_FITC − E3_none] × [E1_both/E1_FITC]) (Doucey et al., 2003; Banga et al., 2020; Song et al., 2020). The different fluorescence values (E) were measured on unlabeled cells (E_none) or cells labeled with FITC (E_FITC) and TRITC (E_TRITC) (Banga et al., 2020; Song et al., 2020).

These techniques can be broadly applied to evaluate intracellular molecular interactions in living cells through pharmacological and molecular studies. They provide a rapid and reliable method of screening molecular interactions, which require standard equipment that are readily available in almost every laboratory.

Part I. Native-PAGE (Polyacrylamide Gel Electrophoresis)