MX1 Antibody

What is MX1 protein and what is its primary biological role?

MX1 (also known as MxA in humans) is an interferon-induced dynamin-like GTPase with potent antiviral activity against a wide range of RNA viruses and some DNA viruses. It functions as a key component of the innate immune system's first line of defense against viral infections.

MX1 has a molecular weight of approximately 75-78 kDa and primarily localizes to the cytoplasm in humans . The protein contains a GTPase domain (amino acids 69-340) and a GTPase Effector Domain (GED) spanning amino acids 574-662 . Its antiviral mechanism involves:

• Binding and inactivating viral ribonucleocapsids of negative-stranded RNA viruses

• Sequestering viral nucleoproteins in perinuclear complexes

• Preventing genome amplification, budding, and viral egress

• Blocking endocytic traffic of incoming virus particles

Importantly, while human MX1 functions in the cytoplasm to block viral replication at stages proximal to transcription, mouse Mx1 acts in the nucleus to inhibit primary viral gene transcription, highlighting important species-specific differences .

How is MX1 expression regulated in human cells?

MX1 expression is primarily induced by type I interferons (particularly IFN-alpha) and type III interferons. When viral RNA or DNA is detected by pattern recognition receptors (PRRs), interferons are produced, leading to MX1 upregulation .

MX1 is strongly induced upon interferon signaling as demonstrated in experimental settings. For example, human peripheral blood mononuclear cells (PBMCs) treated overnight with 20 μg/mL of Recombinant Human IFN-α show significantly increased MX1 expression detectable by western blot . Additionally, viral infections themselves can trigger MX1 expression, though some viruses like HSV-1 may generate truncated 54-57 kDa transcripts with altered function .

Beyond viral infections, MX1 regulation has been implicated in other physiological contexts:

• MX1 is upregulated in response to endoplasmic reticulum stress (ERS)

• Its expression may be altered in certain cancer types, with decreased expression observed in prostate cancer compared to normal tissue

What are the validated applications for MX1 antibodies in research?

MX1 antibodies have been validated for multiple research applications, each with specific optimization requirements:

• Western Blotting (WB): Detects MX1 at approximately 75-78 kDa under reducing conditions. Optimal dilutions range from 1:1000 to 1:10,000 depending on the antibody

• Immunoprecipitation (IP): Typically used at 1:50 dilution

• Immunohistochemistry (IHC-P): Various dilutions (1:100-1:500) are used for paraffin-embedded tissue sections, with recommended heat-induced epitope retrieval

• Immunocytochemistry/Immunofluorescence (ICC/IF): Effective for cellular localization studies at dilutions of approximately 1:100

• Flow Cytometry: Used for intracellular detection of MX1 in single cell suspensions

• ELISA: For quantitative measurement of MX1 in serum and other biological fluids

When selecting an antibody, researchers should consider the target species (human MX1 shares only 49% amino acid sequence identity with mouse Mx1 over amino acids 412-630) , and whether the intended application has been validated by the manufacturer.

For Western Blot:

• Cell lysate preparation: PBMCs or other cell types can be treated with type I interferons (e.g., 20 μg/mL IFN-α) overnight to induce MX1 expression

• Running conditions: Use reducing conditions with appropriate immunoblot buffer systems

• Visualization: HRP-conjugated secondary antibodies followed by chemiluminescent detection yield specific bands at approximately 75 kDa

For Immunohistochemistry:

• Fixation: Formalin/PFA-fixed paraffin-embedded sections are commonly used

• Antigen retrieval: Heat-induced epitope retrieval with basic retrieval reagents is recommended before antibody incubation

• Detection systems: Both fluorescent secondary antibodies and chromogenic detection methods (such as HRP-DAB) have been successfully employed

For Immunofluorescence:

• Cell preparation: Immersion fixation of cells, with particular success reported in IFN-α-treated PBMCs

• Signal enhancement: Counterstaining with DAPI helps visualize nuclear context for the cytoplasmic MX1 signal

• Expected pattern: MX1 shows specific localization to the cytoplasm in human cells

How can I quantitatively measure MX1 protein in biological samples?

For quantitative measurement of MX1 protein in biological fluids such as serum or saliva, ELISA is the method of choice:

• Detection range: Commercial ELISA kits typically have detection ranges of 1.56-100 ng/mL

• Sensitivity: Minimum detectable dose is typically less than 0.65 ng/mL

• Methodology: Double-antibody sandwich ELISA methods are commonly employed

• Sample types: Successfully used for both serum and saliva samples

Key considerations for accurate quantification:

• Regular calibration of pipettes is essential for reliable results

• Standards should be run on each plate for proper quantitation

• Spectrophotometric measurements should be taken within 10 minutes of stopping the reaction

How can MX1 be used as a biomarker in clinical research studies?

MX1 has emerged as a potential biomarker in several clinical contexts:

In Autoimmune Diseases:

• Sjögren's Syndrome: MX1 levels have been associated with disease activity, with research indicating a potential relationship between serum MX1 levels and the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI)

• Clinical correlations: Serum levels of MX1 in patients without Raynaud phenomenon were significantly higher than in those with Raynaud phenomenon (p: 0.029)

• Therapeutic monitoring: Lower MX1 levels have been observed in patients using hydroxychloroquine, suggesting potential utility in monitoring treatment response

In Cancer Research:

• MX1 downregulation has been observed in prostate cancer compared to normal prostate tissue

• Loss of MX1 expression is associated with decreased relapse-free survival in prostate cancer patients

• Positive correlation has been observed between MX1 and HMOX1 (heme oxygenase 1) expression in prostate cancer patients

In Idiopathic Interstitial Pneumonias:

• Anti-MX1 autoantibody levels, particularly IgA, have been identified as significant predictors of prognosis in patients with non-IPF idiopathic interstitial pneumonias

• Multivariate analysis has shown that anti-MX1 IgA antibody levels >0.312 were associated with higher hazard ratios for disease progression (HR: 7.602, 95% CI: 2.013-28.70, p=0.003)

What experimental designs best demonstrate MX1's antiviral mechanisms?

To investigate MX1's antiviral activity, researchers have employed various experimental approaches:

• Viral challenge models: Comparing viral replication kinetics in cells with normal vs. depleted/overexpressed MX1

  • Target viruses include influenza viruses, La Crosse virus (LACV), and thogoto virus (THOV)

• Subcellular localization studies: Using immunofluorescence to track MX1 and viral components

  • MX1 has been shown to sequester viral nucleoproteins in perinuclear complexes

  • Demonstrates the spatial relationship between MX1 and viral replication sites

• Biochemical interaction assays: Investigating direct binding between MX1 and viral components

  • GTPase activity measurements can assess functional aspects of MX1

  • Notably, it is the binding of GTP to MX1, rather than its hydrolysis, that correlates with antiviral activity

• Multimerization studies: Examining the role of MX1 oligomerization in its antiviral function

  • MX1 forms homo-dimers, -tetramers, and -oligomers, with multimerization suggested to be important for activity

Inadequate Induction of MX1 Expression:

MX1 is normally expressed at low levels but is strongly induced by interferons. For positive controls:

• Treat cells with type I interferons (20 μg/mL IFN-α overnight is effective for PBMCs)

• Alternatively, simulate viral infection using poly(I:C) or other TLR agonists

Cross-Reactivity Concerns:

Human MX1 shares only 49% amino acid sequence identity with mouse Mx1 in certain regions . To avoid cross-reactivity issues:

• Carefully select antibodies validated for your species of interest

• Include appropriate negative controls (such as MX1 knockout samples or IFN-receptor knockout cells)

• Consider using recombinant MX1 protein as a positive control for antibody validation

Subcellular Localization Differences:

Human MX1 is predominantly cytoplasmic, while mouse Mx1 is nuclear . When interpreting localization data:

• Be mindful of species-specific differences

• Use appropriate subcellular markers to confirm localization patterns

• Consider that truncated forms of MX1 may show altered localization patterns

For Western Blotting:

• Positive control: Lysates from IFN-α-treated cells (PBMCs or A549 cells are commonly used)

• Negative control: Untreated cells or MX1-knockdown cells

• Loading control: Housekeeping proteins to normalize expression levels

For IHC/ICC:

• Positive tissue control: Human thymus has been validated for MX1 detection

• Antibody controls: Include secondary-only controls to assess background

• Counterstains: DAPI for nuclear visualization helps confirm cytoplasmic localization of MX1

For ELISA:

• Standard curve: Include a full range of standards (typical range: 1.56-100 ng/mL)

• Sample dilutions: Run samples at multiple dilutions to ensure they fall within the linear range

• Technical replicates: Include duplicate or triplicate measurements to assess reproducibility

How does MX1 expression correlate with disease outcomes?

Research has revealed important correlations between MX1 expression and clinical outcomes in several disease contexts:

In Cancer:

• MX1 is among the most consistently downregulated genes in prostate cancer compared to normal prostate tissue

• Loss of MX1 expression is associated with decreased relapse-free survival in prostate cancer patients

• MX1 exhibits a significant positive correlation with HMOX1 (heme oxygenase 1) in prostate cancer patients

In Lung Disease:

• Higher anti-MX1 autoantibody levels, particularly IgA isotype, serve as significant predictors of good prognosis in patients with non-IPF idiopathic interstitial pneumonias

• Multivariate Cox regression analysis showed that anti-MX1 IgA antibody (>0.312) was associated with hazard ratios of 7.602 (95% CI: 2.013-28.70, p=0.003) when adjusted for selected parameters

What is the relationship between MX1 and endoplasmic reticulum stress pathways?

Recent research has uncovered an intriguing relationship between MX1 and endoplasmic reticulum stress (ERS):

• MX1 is upregulated in response to endoplasmic reticulum stress

• ERS can trigger both apoptosis and autophagy in prostate cancer cells

• MX1 silencing has been shown to reverse ERS, suggesting a functional role in stress response pathways

• MX1 enhances ER stress-mediated cell death after influenza virus infection

This relationship suggests a broader role for MX1 beyond direct antiviral activity, potentially connecting innate immunity with cellular stress response pathways.

What are the key differences between monoclonal and polyclonal antibodies against MX1?

When selecting an MX1 antibody, researchers must consider the trade-offs between monoclonal and polyclonal options:

Monoclonal Antibodies:

• Specificity: Examples include rabbit monoclonal antibodies like D3W7I and EPR24485-19 and mouse monoclonal antibodies like clone E-5

• Consistency: Provide superior lot-to-lot consistency and continuous supply

• Applications: Often validated for specific applications like western blot (1:1000), immunoprecipitation (1:50), and IHC (1:160-1:200)

• Epitope targeting: Target specific regions, such as the C-terminal region of MX1

• Available formats: Often available in multiple formats including conjugated versions (HRP, FITC, PE, Alexa Fluor)

Polyclonal Antibodies:

• Recognition: Can recognize multiple epitopes on the MX1 protein

• Examples: Goat anti-human MX1 polyclonal antibodies have been used successfully for detecting MX1 in PBMCs and thymus tissue

• Applications: Often work well across multiple applications including western blot, IHC, and ICC/IF

• Immunogens: Typically raised against recombinant fragments of human MX1, such as amino acids 1-200 or 1-250

The choice depends on research needs: monoclonals offer consistency and specificity for defined epitopes, while polyclonals may provide more robust detection across different applications and sample preparations.

How can I set up experiments to study MX1's role in the interferon response pathway?

To investigate MX1's position in interferon signaling pathways:

• Interferon stimulation experiments:

  • Treat cells with different concentrations of IFN-α (e.g., 20 μg/mL for PBMCs)

  • Include time course analysis (30 minutes to 24 hours) to capture dynamics

  • Measure MX1 protein levels by western blot or immunofluorescence

  • Compare with other interferon-stimulated genes (ISGs) to establish the temporal regulation pattern

• Pathway inhibition studies:

  • Use JAK/STAT pathway inhibitors to confirm the canonical induction mechanism

  • Employ siRNA knockdown of STAT1/STAT2 to assess dependence on these transcription factors

  • Test alternate induction pathways (e.g., via ER stress inducers) to determine if MX1 can be upregulated independently of interferon signaling

• Functional assays:

  • Compare antiviral activity in cells with normal vs. depleted MX1 levels

  • Assess viral replication kinetics using reporter viruses or viral titer measurements

  • Document subcellular distribution patterns using immunofluorescence before and after viral challenge

• Proteomic approaches:

  • Perform immunoprecipitation followed by mass spectrometry to identify MX1-interacting proteins following interferon stimulation

  • Use proximity labeling to identify context-specific interaction networks

These methodological approaches provide complementary data on MX1's regulation and function within the interferon response pathway.