Interferon regulatory factor 3 (IRF3)

Structure of IRF3 Target Protein

Figure 1: Structure of IRF3 Target Protein.

Introduction to IRF3

Protein Function

IRF3 (Interferon regulatory factor 3) is a member of the interferon regulatory factor family and plays a crucial role in the innate immune response against viruses . IRF3 regulates the expression of interferons (IFNs) and IFN-stimulated genes (ISG).
The interferon regulatory factor family includes 9 members. IRF3 is a key transcription factor in the cellular antiviral response and is expressed constitutively in variety of tissues.

Protein Characteristics

In resting cells, IRF3 is located in the cytoplasm maintained as a monomer in an autoinhibited state.
Upon viral infection or other stimuli, phosphorylated IRF3 then undergoes dimerization, leading to the translocation of IRF3 dimers from the cytoplasm to the nucleus, where IRF3 activates the expression of type I IFNs and other pro-inflammatory cytokines.

Protein Expression

IRF3 is constitutively expressed in a variety of tissues.
Phosphorylated IRF3 (p-IRF3) is typically detectable only after induction.

Protein Localization

IRF3 is primarily located in the cytoplasm and nucleus. It resides in the cytoplasm when inactive and translocate to the nucleus upon activation.

IRF3 ICC Experimental Results Image, Recombinant Anti-IRF3 Antibody [EPR2418Y] (ab68481)

Figure 2: IRF3 ICC Experimental Results Image, Recombinant Anti-IRF3 Antibody [EPR2418Y] (ab68481)

Sample: HeLa cell line.
Experimental Conditions: Fixed with 4% PFA, permeabilized with 0.1% Triton X-100.
Green: IRF3, Red: Tubulin, Blue: DAPI.

Isoforms & Post-translational Modifications

Human (Q14653): Isoforms 1, 2, and 4: 33-49 kDa (predicted);
Isoforms 3 and 5: 12-17 kDa (predicted)
Mouse (P70671): 47 kDa (predicted)
IRF3 undergoes phosphorylation.
IRF3 undergoes ubiquitination.
Phosphorylated IRF3 can dimerize and translocate to the nucleus.

Tips for WB Experiment

Precautions

IRF3 undergoes phosphorylation, and detecting p-IRF3 typically requires sample induction (e.g., Calyculin A treatment for 45 minutes in MCF7 cells).
When detecting phosphorylated modifications, ensure the total amount of IRF3 protein in the cells.
Use freshly prepared sample lysates to prevent protein degradation due to freezing and add appropriate phosphatase inhibitors.
Use freshly prepared antibodies; reusing antibodies is not recommended.

Positive Controls

IRF3: HeLa, MCF7.
p-IRF3: - MCF7 treated with Calyculin A.

Example Results

Recombinant Anti-IRF3 Antibody [EPR2418Y] (ab68481)

Figure 3: Recombinant Anti-IRF3 Antibody [EPR2418Y] (ab68481)

Lane 1: Jurkat whole cell lysate
Lane 2: MCF7 whole cell lysate
Lane 3: Wild-type HeLa whole cell lysate
Lane 4: IRF3 knockout HeLa whole cell lysate

Predicted band size: 47 kDa
Observed band size: 50 kDa, Green: IRF3, Red: GAPDH

Recombinant Anti-IRF3 (phospho S386) Antibody [EPR2346] (ab76493)

Figure 4: Recombinant Anti-IRF3 (phospho S386) Antibody [EPR2346] (ab76493)

Lane 1 : MCF7 cell lysate - treated with Calyculin A
Lane 2 : MCF7 cell lysate – untreatedPredicted band size: 47 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

Add a protease inhibitor cocktail to prevent degradation of target proteins.
Keep samples on ice throughout the entire sample preparation process.
Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

Load at least 20μg total protein for electrophoresis.

Transfer:

It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.

Antibody incubation:

Select a suitable antibody working concentration according to the product datasheet.
It is recommended to dilute the primary and secondary antibodies with blocking buffer.