NOXA1 Antibody

What is NOXA1 and why is it significant for research?

NOXA1 (NADPH oxidase activator 1) functions as an important regulatory component in the assembly and activation of the NADPH oxidase complex . It serves as an activator of NOX1, a superoxide-producing NADPH oxidase, and plays a critical role in the production of reactive oxygen species (ROS) . These ROS participate in numerous biological processes including host defense, hormone biosynthesis, oxygen sensing, and signal transduction pathways . Research has established that NOXA1 is the functional homolog of p67phox in vascular smooth muscle cells (VSMCs), where it regulates redox signaling and VSMC phenotype . This makes NOXA1 an important research target for vascular pathologies including atherosclerosis and neointimal hyperplasia.

What are the key applications for NOXA1 antibodies in experimental research?

NOXA1 antibodies have been validated for multiple research applications, allowing for comprehensive protein analysis. The primary applications include:

• Western blot (WB) - For detecting and quantifying NOXA1 protein expression in cell or tissue lysates (typical dilution ranges from 1:1000-1:5000)

• Immunohistochemistry (IHC) - For visualizing the distribution and localization of NOXA1 in tissue sections (recommended dilution 1:20-1:200)

• Immunocytochemistry/Immunofluorescence (ICC/IF) - For detecting NOXA1 in cultured cells (recommended dilution 1:50-1:200)

• ELISA - For quantitative analysis of NOXA1 protein levels

These applications enable researchers to investigate NOXA1 expression patterns, cellular localization, protein interactions, and functional roles in various experimental contexts.

How do I select the appropriate NOXA1 antibody for my specific research model?

When selecting a NOXA1 antibody, consider the following factors based on your research model:

• Species reactivity: Confirm the antibody reacts with your experimental model species. Commercial NOXA1 antibodies are available with reactivity to human and mouse samples .

• Clonality: Both polyclonal (rabbit and mouse) options are available :

  • Rabbit polyclonal antibodies recognize epitopes within human NOXA1 aa 150-350 region

  • Mouse polyclonal antibodies against full-length human NOXA1 protein

• Application validation: Verify the antibody has been validated for your specific application. For example, ab222852 is suitable for WB, IHC-P, and ICC/IF applications , while CSB-PA015963LA01HU is validated for ELISA, WB, IHC, and IF .

• Positive controls: Use appropriate positive controls. For Western blot, mouse brain lysate (for rabbit polyclonal antibody) or NOXA1-transfected 293T lysate (for mouse polyclonal antibody) have been used successfully.

• Conjugation requirements: Determine if you need a conjugated antibody (HRP, FITC, or biotin) for specific applications such as ELISA or direct fluorescence imaging .

How can NOXA1 antibodies be used to investigate the role of NOXA1 in vascular pathologies?

NOXA1 has been identified as a significant regulator in vascular smooth muscle cell (VSMC) function and vascular disease progression. To investigate its role using NOXA1 antibodies:

• Expression analysis in disease models: NOXA1 expression is significantly increased in aortas and atherosclerotic lesions of ApoE-/- mice compared to age-matched wild-type mice . IHC with NOXA1 antibodies can be used to compare expression between normal and diseased vessels.

• Localization studies: Immunohistochemical analysis has revealed that unlike p67phox, immunoreactive NOXA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions . This makes NOXA1 antibodies valuable tools for studying disease progression.

• Protein interaction studies: Co-immunoprecipitation with NOXA1 antibodies can detect the interaction between NOXA1 and p47phox, which increases after thrombin stimulation . This approach can reveal how NOXA1 assembles with other NADPH oxidase components during activation.

• Intervention studies: NOXA1 antibodies can be used to evaluate the effectiveness of interventions targeting NOXA1. Research has shown that suppression of NOXA1 expression decreases thrombin-induced superoxide production, while overexpression increases it , supporting the potential for therapeutic targeting of NOXA1.

What protocols are effective for investigating NOXA1-p47phox interactions using NOXA1 antibodies?

To investigate NOXA1-p47phox interactions, which are critical for NADPH oxidase function, the following immunoprecipitation protocol has been effective:

• Expression system preparation: If endogenous protein levels are low, transfect cells (such as p47phox-deficient VSMCs) with tagged constructs (e.g., HA-tagged NOXA1 and myc-tagged p47phox) .

• Stimulation: Treat cells with or without agonists like thrombin (10 minutes) to induce complex formation .

• Immunoprecipitation procedure:

  • Prepare cell lysates under non-denaturing conditions

  • Perform immunoprecipitation using anti-HA antibody (for HA-tagged NOXA1)

  • Analyze the immunoprecipitates by Western blotting with anti-myc antibody (for myc-tagged p47phox)

This protocol has successfully demonstrated that thrombin stimulation significantly increases the interaction between NOXA1 and p47phox , suggesting enhanced NADPH oxidase complex formation upon agonist stimulation.

How can I validate NOXA1 antibody specificity in my experimental system?

Validating antibody specificity is crucial for reliable results. For NOXA1 antibodies, consider these approaches:

• Genetic validation:

  • Use cells with modulated NOXA1 expression (overexpression or knockdown)

  • Retroviral or adenoviral delivery of NoxA1-specific shRNA has been shown to decrease NOXA1 expression by approximately 90%

  • Western blot should show corresponding changes in the 51-kDa band recognized by the NOXA1 antibody

• Band size verification:

  • Confirm the observed band matches the predicted molecular weight of NOXA1 (51 kDa)

  • Note that some antibodies may detect NOXA1 at slightly higher apparent molecular weights (e.g., 60 kDa for ab68523) , which could be due to post-translational modifications

• Control experiments:

  • Include positive controls such as NOXA1-transfected 293T lysate

  • Use non-transfected lysates as negative controls

  • For mouse samples, mouse brain lysate has been validated as an appropriate positive control

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • This should abolish specific staining or bands if the antibody is specific

What are the optimal conditions for Western blot detection of NOXA1?

For optimal Western blot detection of NOXA1 protein, the following conditions have been successfully used:

• Sample preparation:

  • Mouse brain lysate for detection of endogenous NOXA1

  • NOXA1-transfected 293T lysate for overexpression systems

  • Vascular smooth muscle cell lysates for vascular research

• Antibody dilutions:

  • Anti-NOXA1 rabbit polyclonal antibody (ab222852): 1/1000 dilution

  • Anti-NOXA1 mouse polyclonal antibody (ab68523): 1/500 dilution

  • Boster NOXA1 antibody (PA1930): 0.1-0.5μg/ml for human samples

• Secondary antibody selection:

  • For rabbit primary: Goat polyclonal to rabbit at 1/10000 dilution

  • For mouse primary: Goat Anti-Mouse IgG (H&L)-HRP at 1/2500 dilution

• Expected results:

  • Predicted band size: 51 kDa

  • Some antibodies may detect bands at slightly different sizes (e.g., 60 kDa)

  • Validated positive controls include human U87 whole cell or human HeLa whole cell lysates

How should I optimize immunohistochemistry protocols for NOXA1 detection in tissue samples?

For effective IHC detection of NOXA1 in tissue samples:

• Tissue preparation:

  • Paraffin embedding has been validated for human lung cancer tissue

  • For vascular tissues, standard fixation and paraffin embedding protocols are suitable for detecting NOXA1 in vessel walls

• Antibody conditions:

  • Rabbit polyclonal anti-NOXA1 (ab222852) has been used at 1/100 dilution for paraffin-embedded tissues

  • For research-grade antibodies like CSB-PA015963LA01HU, dilutions between 1:20-1:200 are recommended

• Antigen retrieval:

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0) is typically effective for NOXA1 detection

  • Optimization may be needed depending on tissue type and fixation conditions

• Expected staining patterns:

  • Cytoplasmic staining is expected as NOXA1 is primarily localized in the cytoplasm of cells

  • In vascular tissues, look for staining in both medial and intimal smooth muscle cells

  • In atherosclerotic lesions, NOXA1 immunoreactivity should be observable in intimal and medial SMCs

• Controls:

  • Include known positive tissues (e.g., lung cancer samples)

  • Include negative controls by omitting primary antibody

What approach should I use to monitor NOXA1-dependent ROS production with NOXA1 antibodies?

To investigate NOXA1-dependent ROS production using NOXA1 antibodies in combination with ROS detection methods:

• Experimental design:

  • Modulate NOXA1 expression using:

    • Retroviral NOXA1-specific shRNA for knockdown (90% reduction)

    • Adenoviral NoxA1 for overexpression

  • Verify expression changes by Western blot with NOXA1 antibodies

• ROS detection methods:

  • Lucigenin-enhanced chemiluminescence assay for superoxide measurement

  • Dihydroethidium (DHE) to ethidium conversion for superoxide detection in intact cells

  • DCF fluorescence for general ROS detection

• Experimental validation:

  • In wild-type VSMCs, thrombin treatment (10 minutes) significantly increases ROS production

  • This increase is markedly attenuated in cells expressing NoxA1 shRNA

  • NoxA1 shRNA does not completely inhibit ROS production, suggesting low levels of NoxA1 or other mechanisms contribute to basal ROS production

• Controls:

  • Use scrambled shRNA as negative control for knockdown studies

  • Verify that manipulation of NOXA1 expression doesn't affect Nox1 expression using real-time RT-PCR

  • Check for absence of cellular toxicity or stress response activation (e.g., by measuring HSP70 levels)

How can NOXA1 antibodies be used to investigate NADPH oxidase complex assembly dynamics?

NOXA1 antibodies can provide insights into the dynamic assembly of NADPH oxidase complexes:

• Translocation studies:

  • NOXA1 is expressed in the cytoplasm of VSMCs and translocates to the cell membrane upon stimulation

  • Use NOXA1 antibodies for immunofluorescence to track translocation after stimulation with agonists like epidermal growth factor

  • Co-staining with p47phox can reveal co-translocation patterns

• Protein interaction networks:

  • Immunoprecipitation with NOXA1 antibodies followed by mass spectrometry can identify novel interaction partners

  • This approach can be used to compare interaction networks under basal and stimulated conditions

• Live cell imaging:

  • For dynamic studies, express fluorescently-tagged NOXA1 constructs

  • Validate expression and functionality using NOXA1 antibodies before imaging experiments

  • Measure the kinetics of complex assembly in real-time

• Temporal activation patterns:

  • Use NOXA1 antibodies to determine the timeline of complex assembly after stimulation

  • Compare with the kinetics of ROS production to establish cause-effect relationships

What are the considerations for using NOXA1 antibodies in studying the role of NOXA1 in redox-sensitive protein kinase activation?

NOXA1 has been implicated in the activation of redox-sensitive protein kinases. To study this role:

• Experimental approach:

  • Modulate NOXA1 expression using shRNA or overexpression systems

  • Stimulate cells with agonists like thrombin

  • Use phospho-specific antibodies to detect activation of kinases alongside NOXA1 antibodies for expression verification

• Key kinases to examine:

  • Janus kinase 2 (JAK2)

  • Akt

  • p38 mitogen-activated protein kinase (MAPK)

• Expected outcomes:

  • Infection with NoxA1 shRNA significantly decreases thrombin-induced activation of these redox-sensitive protein kinases in VSMCs

  • This suggests NOXA1-dependent ROS production is essential for these signaling pathways

• Methodological considerations:

  • Include time course experiments to determine activation kinetics

  • Use antioxidants as controls to confirm ROS-dependence

  • Consider the use of specific inhibitors for each kinase to establish pathway hierarchies

How can NOXA1 antibodies contribute to translational research targeting vascular diseases?

NOXA1 antibodies can significantly advance translational research on vascular diseases:

• Biomarker potential:

  • NoxA1 expression is significantly increased in aortas and atherosclerotic lesions of ApoE-/- mice compared with age-matched wild-type mice

  • Use NOXA1 antibodies to quantify expression levels in patient samples for potential diagnostic applications

• Therapeutic target validation:

  • Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increases superoxide production in medial VSMCs and enhances neointimal hyperplasia

  • NOXA1 antibodies can be used to confirm target engagement of NOXA1-directed therapeutics

• Patient stratification:

  • Unlike p67phox, immunoreactive NOXA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions

  • This differential expression pattern could be exploited for patient stratification using NOXA1 antibodies

• Preclinical to clinical translation:

  • Use NOXA1 antibodies to verify consistency of expression and function between animal models and human samples

  • Develop standardized immunohistochemical protocols for potential clinical applications