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Updated: Nov 1, 2025
Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization
Published on: October 6, 2019
Intracellular IRF5 Dimerization Assay
Cherrie D Sherman1, Betsy J Barnes1
1Center for Autoimmune Musculoskeletal and Hematopoietic Diseases, Feinstein Institutes for Medical Research, Manhasset, New York, USA.
Insights
This study introduces two assays to detect interferon regulatory factor 5 (IRF5) homodimerization and interactions with cell-penetrating peptide (CPP) inhibitors. These methods offer rapid and accurate detection of IRF5 molecular interactions in cells.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Interferon regulatory factor 5 (IRF5) plays a crucial role in innate immunity and inflammatory responses.
- Understanding IRF5's molecular interactions, such as homodimerization and binding to inhibitors, is vital for deciphering its regulatory mechanisms.
- Existing methods for studying endogenous IRF5 interactions are limited.
Purpose of the Study:
- To develop and validate novel assays for detecting endogenous IRF5 homodimerization.
- To establish a method for assessing the interaction between endogenous IRF5 and cell-penetrating peptide (CPP) inhibitors.
- To provide tools for rapid and accurate analysis of IRF5 molecular interactions.
Main Methods:
- Native-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting to detect IRF5 homodimers in THP-1 cells treated with an IRF5-targeted CPP (IRF5-CPP) inhibitor.
- In-cell fluorescence resonance energy transfer (FRET) assay to measure the interaction between endogenous IRF5 and FITC-labeled IRF5-CPP in THP-1 cells.
- Stimulation of THP-1 cells with R848 to activate IRF5 signaling pathways.
Main Results:
- Successfully detected endogenous IRF5 homodimers using native-PAGE and immunoblotting.
- Demonstrated the ability to measure IRF5-CPP interactions using an in-cell FRET assay.
- Quantified fluorescence transfer as FRET units, indicating successful molecular interaction detection.
Conclusions:
- The developed native-PAGE and FRET assays are effective for detecting endogenous IRF5 homodimerization and IRF5-CPP interactions.
- These assays provide rapid, accurate, and robust methods for studying IRF5 molecular interactions.
- The findings facilitate further research into IRF5 function and the development of targeted therapeutics.
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.
