Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

Summary

A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

Abstract

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like receptor myeloid differentiation primary response gene 88 (TLR-MyD88) signaling pathway. IRF5 activation involves phosphorylation, dimerization, and subsequent translocation from the cytoplasm into the nucleus, which in turn induces the gene expression of various pro-inflammatory cytokines. A detection assay for IRF5 activation is essential to studying IRF5 functions and its relevant pathways. This article describes a robust assay to detect endogenous IRF5 activation in the CAL-1 human plasmacytoid dendritic cell (pDC) line. The protocol consists of a modified nondenaturing electrophoresis assay that can distinguish IRF5 in its monomer and dimer forms, thus providing an affordable and sensitive approach to analyze IRF5 activation.

Introduction

Interferon regulatory factor 5 (IRF5) is an important transcription regulator that plays a prominent role in regulating the immune response, particularly in the release of pro-inflammatory cytokines and type I interferons (IFNs)^1,2,3. Misregulation of IRF5 is a contributing factor in numerous autoimmune diseases, as evident by various polymorphisms in the IRF5 locus that are associated with systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, etc.^4,5,6,<....

Protocol

NOTE: The protocol described here uses CAL-1 pDC cell line treated with resiquimod (R848), an agonist for TLR7/8. This protocol has been applied to other human and murine cell types, including RAW 264.7 (murine macrophage line), THP-1 (human monocytic cell line), BJAB (human B cell line), Ramos (human B cell line), and MUTZ-3 (human dendritic cell line)²³.

1. Stimulation of CAL-1 Cells

1. Maintain CAL-1 cell cultures in a T75 flask at 37 °C and 5% CO₂ under sterile conditions with 20−25 mL of RPMI 1640 medium containing 5% fetal bovine serum (FBS), 25 mM HEPES, and 1x m....

Results

The immunoblot (IB) with an anti-IRF5 antibody was performed on CAL-1 cells unstimulated or stimulated with 1 µg/mL R848 for 2 h (Figure 1). Cell lysates were prepared, and the native PAGE was performed. In unstimulated CAL-1 cells, IRF5 was detected as a single band on the native PAGE, corresponding to its monomeric form. Upon treatment of CAL-1 cells with R848 for 2 h, the level of IRF5 monomer decreased with a concurrent increase in the accumulation of a slowly migrating band that corresp.......

Discussion

The protocol described here is a modified native PAGE that distinguishes both monomeric and dimeric forms of endogenous IRF5. There have been few studies reporting the detection of endogenous IRF5 activation using the specialized imaging flow cytometry technique^23,27,28,30. This protocol uses a common technique and commonplace reagents and tools to assess the endogenous IRF5 activation state du.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Life Technologies, HK	21985023
300 W/250 V power supply 230 V AC	Life Technologies, HK	PS0301
Anti-IRF5 antibody	Bethyl Laboratories, USA	A303-385
BIOSAN Rocker Shaker (cold room safe)	EcoLife, HK	MR-12
EDTA Buffer, pH 8, 0.5 M 4x 100 mL	Life Technologies	15575020
Glycerol 500 mL	Life Technologies	15514011
Glycine	Life Technologies, HK	15527013
Goat anti-Mouse IgG DyLight 800 Conjugated Antibody	LAB-A-PORTER/Rockland, HK	610-145-002-0.5
Goat anti-Rabbit IgG DyLight 800 Conjugated Antibody	LAB-A-PORTER/Rockland, HK	611-145-002-0.5
Halt protease inhibitor cocktail (100x)	Thermo Fisher Scientific, HK	78430
HEPES	Life Technologies, HK	15630080
LI-COR Odyssey Blocking Buffer (TBS)	Gene Company, HK	927-50000
Mini Tank blot module combo; Transfer module, accessories	Life Technologies, HK	NW2000
NativePAGE 3-12% gels, 10 well kit	Life Technologies, HK	BN1001BOX
NativePAGE Running Buffer 20x	Life Technologies, HK	BN2001
NativePAGE Sample Buffer 4x	Life Technologies, HK	BN2003
NP-40 Alternative, Nonylphenyl Polyethylene Glycol	Tin Hang/Calbiochem, HK	#492016-100ML
PBS 7.4	Life Technologies, HK	10010023
Polyvinylidene difluoride (PVDF) membrane	Bio-gene/Merck Millipore, HK	IPFL00010
Protein assay kit II (BSA)	Bio-Rad, HK	5000002
R848	Invivogen, HK	tlrl-r848
RPMI 1640	Life Technologies, HK	61870127
Sodium Chloride	ThermoFisher	BP358-1
Sodium deoxycholate ≥97% (titration)	Tin Hang/Sigma, HK	D6750-100G
Tris	Life Technologies, HK	15504020
TWEEN 20	Tin Hang/Sigma, HK	#P9416-100ML