AZIN1 Antibody

What is AZIN1 and what cellular functions does it regulate?

AZIN1 (Antizyme inhibitor 1) is a protein that positively regulates ornithine decarboxylase (ODC) activity and polyamine uptake. It functions as an enzymatically inactive ODC homolog that counteracts the negative effects of ODC antizymes (AZs) OAZ1, OAZ2, and OAZ3 on ODC activity. AZIN1 accomplishes this by competing with ODC for antizyme-binding, thereby inhibiting antizyme-dependent ODC degradation and releasing ODC monomers from their inactive complex with antizymes. This process leads to the formation of catalytically active ODC homodimers and restores polyamine production .

Beyond its role in polyamine regulation, AZIN1 has been identified as playing an active role in cell proliferation through its interaction with cyclin D1. By preventing cyclin D1 degradation, AZIN1 extends the half-life of this critical cell cycle regulator . AZIN1 expression notably increases during specific phases of the cell cycle, particularly in G1 and G2/M phases, indicating its temporal importance in cell division processes .

What are the optimal applications for AZIN1 antibodies in cancer research?

AZIN1 antibodies are particularly valuable in cancer research due to AZIN1's established role in multiple malignancies. The primary applications include:

• Immunohistochemistry (IHC): Used for examining AZIN1 expression patterns in tumor sections compared to normal tissues. This application is especially important since AZIN1 is upregulated in multiple cancers and drives cancer progression through dysregulation of polyamine synthesis and cell-cycle control . Recommended dilution for IHC applications is 1:50-400 based on the specific antibody .

• Western blotting (WB): Essential for quantifying AZIN1 protein levels and validating RNA editing status in tumor samples. Typically used at dilutions of 1:500-2000 .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Valuable for determining subcellular localization of AZIN1, particularly important since RNA-edited AZIN1 can translocate from the cytoplasm to the nucleus in cancer cells . Optimal working dilutions range from 1:50-400 .

• ELISA: Useful for quantitative measurement of AZIN1 in research samples at dilutions of 1:100-1000 .

• Co-immunoprecipitation: Important for studying AZIN1 interactions with binding partners such as OAZ1, OAZ2, and cyclin D1 in cancer models.

How can I differentiate between wild-type and RNA-edited AZIN1 in experimental settings?

Differentiating between wild-type and RNA-edited AZIN1 requires a multi-faceted approach:

• Sanger sequencing validation: The gold standard for confirming RNA editing at the AZIN1 S367G site. As demonstrated in NSCLC research, this method can verify the conversion of the genomically encoded serine (AGT) to glycine (GGT) at the edited site .

• Quantitative PCR: For relative quantification of wild-type versus edited AZIN1 transcripts. This approach was successfully employed in studies examining AZIN1 editing in NSCLC cell lines .

• Custom antibodies: Though not widely available, specialized antibodies designed to recognize the conformational change resulting from S367G substitution can be developed for direct differentiation.

• Functional assays: Since edited AZIN1 exhibits altered binding affinity to antizymes, binding assays that measure differential interaction with OAZ1 can indirectly indicate editing status. The edited form typically shows enhanced stability and altered subcellular distribution .

• Subcellular localization: Immunofluorescence can detect the characteristic shift of edited AZIN1 from cytoplasmic to nuclear localization, which serves as a visual indicator of the editing event .

What are the optimal fixation and permeabilization methods for AZIN1 immunofluorescence studies?

For optimal visualization of AZIN1 in immunofluorescence studies, the following protocol has been validated in cancer cell research:

• Fixation: Fix cells in 100% methanol for 10 minutes at room temperature. This fixation method preserves AZIN1 epitopes while maintaining cellular architecture .

• Permeabilization: Permeate with 0.5% Triton X-100 in PBS for 10 minutes to allow antibody access to intracellular compartments .

• Blocking: Use 10% BSA in 0.2% PBS-Tween for 30 minutes at 37°C to reduce non-specific binding .

• Primary antibody incubation: Apply AZIN1 antibody (such as ab57169 from Abcam) at a 1/100 dilution and incubate overnight at 4°C for optimal binding .

• Secondary antibody application: Use fluorochrome-conjugated secondary antibodies such as Alexa Fluor® 594 goat anti-mouse IgG at a 1/1000 dilution for 45 minutes at room temperature .

• Nuclear counterstaining: Apply ProLong® Gold Antifade Reagent with DAPI to visualize nuclei, which is crucial for determining nuclear translocation of edited AZIN1 .

This protocol is particularly effective for observing the subcellular redistribution of AZIN1 that occurs with RNA editing.

How can I analyze AZIN1 interaction with OAZ1 and ODC using co-immunoprecipitation techniques?

Analyzing AZIN1 interactions with OAZ1 and ODC requires careful co-immunoprecipitation approaches:

• Antibody selection: Use high-affinity antibodies against AZIN1 that do not interfere with the AZBE (Antizyme-Binding Element) region. This is critical since AZIN1-OAZ1 binding (Kd = 20 nM) is approximately 10-fold stronger than ODC-OAZ1 interaction (Kd = 200 nM) .

• Lysis conditions: Use mild lysis buffers containing 1% NP-40 or 0.5% Triton X-100 to preserve protein-protein interactions. Avoid harsh detergents like SDS that would disrupt these interactions.

• Pre-clearing: To reduce non-specific binding, pre-clear lysates with protein A/G beads before immunoprecipitation.

• Controls: Include:

  • IgG control to assess non-specific binding

  • Input control (10% of lysate) to confirm protein expression

  • Reciprocal IP (using OAZ1 or ODC antibodies) to validate interactions

• Binding analysis: After co-immunoprecipitation, analyze samples by western blotting to detect OAZ1 and ODC pulled down with AZIN1. For quantitative analysis, compare wild-type AZIN1 with edited AZIN1 (S367G) to determine if RNA editing affects binding dynamics .

• Alternative approach: Consider proximity ligation assays (PLA) as a complementary method to visualize and quantify AZIN1-OAZ1 interactions in situ.

What experimental designs can differentiate between polyamine-dependent and independent functions of AZIN1?

Distinguishing between polyamine-dependent and independent functions of AZIN1 requires sophisticated experimental designs:

• Polyamine depletion/supplementation experiments:

  • Deplete cellular polyamines using DFMO (difluoromethylornithine), an ODC inhibitor

  • Supplement media with exogenous polyamines (putrescine, spermidine, spermine)

  • Compare AZIN1-mediated effects under both conditions

• AZIN1 mutant constructs:

  • Generate AZIN1 mutants with altered OAZ binding (K125N and K140M) that show decreased binding to OAZ1 by 10-fold

  • Compare these with wild-type AZIN1 in functional assays

  • Effects that persist with binding-deficient mutants suggest polyamine-independent functions

• Cyclin D1 stability assays:

  • Measure cyclin D1 half-life in cells expressing wild-type AZIN1, edited AZIN1, or AZIN1 knockdown

  • Perform these experiments with and without polyamine depletion

  • Effects on cyclin D1 that persist despite polyamine depletion indicate polyamine-independent regulation

• Cell cycle analysis:

  • Track AZIN1 subcellular localization throughout the cell cycle using time-lapse microscopy

  • Correlate with polyamine levels measured by HPLC or LC-MS

  • Determine if AZIN1 colocalization with OAZ1 at centrosomes during prophase to late anaphase is polyamine-dependent

What controls should be included when studying AZIN1's role in angiogenesis via IL-8 upregulation?

When investigating AZIN1's role in angiogenesis through IL-8 upregulation, several critical controls must be included:

• AZIN1 expression controls:

  • Wild-type AZIN1 overexpression

  • RNA-edited AZIN1 (S367G) overexpression

  • AZIN1 knockdown using siRNA or shRNA

  • Empty vector control

• IL-8 pathway controls:

  • IL-8 neutralizing antibodies to confirm specificity

  • IL-8 receptor antagonists (such as reparixin) to validate downstream signaling

  • Recombinant IL-8 supplementation to rescue phenotypes

• c-Myc degradation pathway controls:

  • OAZ2 knockdown to confirm involvement in the ubiquitin-independent proteasome pathway

  • Proteasome inhibitors to verify degradation mechanism

  • c-Myc mutants resistant to OAZ2-mediated degradation

• Angiogenesis assay controls:

  • Positive controls: VEGF-treated cells/tissues

  • Negative controls: Angiogenesis inhibitors (e.g., bevacizumab)

  • Endothelial cell-specific markers (CD31, vWF) for quantification

• In vivo controls:

  • Age/sex-matched animals

  • Multiple xenograft sites to account for microenvironment variation

  • Contralateral injection of control cells within the same animal to minimize inter-animal variability

What are the recommended approaches for correlating AZIN1 protein levels with RNA editing status in tumor samples?

Correlating AZIN1 protein levels with RNA editing status in tumor samples requires an integrated approach:

• RNA editing quantification:

  • RNA-seq with sufficient depth to detect editing events

  • Site-specific qRT-PCR using primers that discriminate between edited and unedited transcripts

  • Sanger sequencing to confirm editing at position S367G

• Protein level assessment:

  • Western blotting with calibrated loading controls

  • Immunohistochemistry with quantitative image analysis

  • ELISA for high-throughput quantification

• Correlation analysis methods:

  • Calculate editing index (percentage of edited transcripts)

  • Plot protein levels against editing index

  • Perform regression analysis to determine relationship strength

• Multi-parameter analysis:

  • Integrate editing status with protein level, subcellular localization, and functional outcomes

  • Consider creating a composite score that incorporates all parameters

• Clinical sample considerations:

  • Use matched tumor/normal samples from the same patient

  • Account for tumor heterogeneity by sampling multiple regions

  • Consider the 20% incidence rate of AZIN1 S367G editing observed in NSCLC samples as a baseline reference

How can I validate antibody specificity for AZIN1 in different experimental contexts?

Thorough validation of AZIN1 antibody specificity is essential for reliable research outcomes:

• Western blot validation:

  • Confirm single band at expected molecular weight (~49.5 kDa)

  • Include positive controls: Tissue/cells known to express AZIN1

  • Include negative controls: AZIN1 knockdown samples

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide

• Immunohistochemistry/Immunocytochemistry validation:

  • Compare staining patterns with published literature

  • Include isotype control antibody

  • Test specificity on tissues from knockout models if available

  • Demonstrate reduced staining after AZIN1 siRNA treatment

• Cross-reactivity assessment:

  • Test against purified AZIN2 protein or AZIN2-overexpressing cells

  • Check for cross-reactivity with ODC due to structural similarities

  • Verify specificity in multiple species if antibody claims multi-species reactivity

• Functional validation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Confirm antibody can detect both wild-type and edited AZIN1 forms

  • Verify detection of induced overexpression in transfection experiments

• Documentation requirements:

  • Record antibody dilutions tested (e.g., 1:50-400 for IHC, 1:500-2000 for WB)

  • Document all validation methods in publications

  • Note batch/lot numbers as antibody performance can vary between lots

What are the recommended positive control tissues or cell lines for AZIN1 antibody validation?

Based on research findings, the following positive controls are recommended for AZIN1 antibody validation:

• Cell lines:

  • A549 and H1299 (NSCLC lines): Demonstrate detectable AZIN1 expression and have been successfully used in AZIN1 overexpression studies

  • HEK293: Express moderate levels of endogenous AZIN1

  • HepG2: Liver cancer cells with documented AZIN1 RNA editing

• Tissue samples:

  • Liver tissue: Shows consistent AZIN1 expression

  • Prostate tissue: AZIN1 is substantially elevated in prostate cancer

  • Brain tissue: Exhibits AZIN1 upregulation in cancer states

  • Breast tissue: Demonstrates differential expression between normal and cancer samples

• Transfection controls:

  • GFP-labeled wild-type AZIN1 expression constructs

  • GFP-labeled edited AZIN1 expression constructs

• Animal models:

  • NOD-scid-IL2Rg−/− (NSI) mice with A549 AZIN1-edited xenografts have been validated for in vivo studies

How should I design experiments to study AZIN1's role in cell cycle regulation?

When investigating AZIN1's role in cell cycle regulation, consider these experimental design principles:

• Cell synchronization approaches:

  • Double thymidine block for G1/S synchronization

  • Nocodazole treatment for G2/M arrest

  • Serum starvation for G0/G1 arrest

• AZIN1 expression monitoring:

  • Time-course analysis following synchronization release

  • Quantitative western blotting at 2-hour intervals

  • Immunofluorescence to track subcellular localization changes

  • Flow cytometry to correlate AZIN1 levels with cell cycle phases

• Centrosome colocalization studies:

  • Dual immunofluorescence for AZIN1 and OAZ1

  • Co-staining with centrosome markers (γ-tubulin)

  • Track colocalization from prophase to late anaphase

• Cyclin D1 stability assessment:

  • Cycloheximide chase experiments in AZIN1 knockdown vs. control cells

  • Pulse-chase labeling to measure cyclin D1 half-life

  • Proteasome inhibitor controls to confirm degradation pathway

• Cell proliferation assays:

  • Compare wild-type AZIN1, edited AZIN1, and AZIN1 knockdown

  • Monitor using real-time cell analysis systems

  • Correlate with polyamine measurements to distinguish mechanisms

• Critical controls:

  • AZIN1 mutants with altered OAZ binding capacity

  • Cyclin D1 mutants resistant to degradation

  • OAZ1/OAZ2 knockdown to assess antizyme dependency

How can AZIN1 antibodies be utilized to study the cancer angiogenesis mechanism?

AZIN1 antibodies offer powerful tools for investigating the newly discovered role of AZIN1 in cancer angiogenesis:

• Mechanistic pathway analysis:

  • Use AZIN1 antibodies in chromatin immunoprecipitation (ChIP) assays to investigate edited AZIN1's potential direct or indirect regulation of IL-8 transcription

  • Combine with RNA-seq to identify global transcriptional changes mediated by RNA-edited AZIN1

• Protein complex identification:

  • Employ immunoprecipitation with AZIN1 antibodies followed by mass spectrometry to identify novel interacting partners involved in angiogenesis

  • Validate interactions using proximity ligation assays in tumor tissues

• Tumor microenvironment analysis:

  • Perform multiplex immunofluorescence with AZIN1, IL-8, and endothelial cell markers (CD31) in tumor sections

  • Quantify microvascular density in relation to AZIN1 expression/editing status

  • Correlate with infiltrating immune cells to assess broader microenvironment effects

• Therapeutic response monitoring:

  • Use AZIN1 antibodies to track changes in expression/localization following treatment with angiogenesis inhibitors or IL-8 receptor antagonists like reparixin

  • Develop IHC protocols to assess AZIN1 as a biomarker for anti-angiogenic therapy response

• In vivo experimental design:

  • Create xenograft models with differential AZIN1 expression/editing status

  • Perform immunohistochemistry on harvested tumors to correlate AZIN1 levels with vascular parameters

  • Use window chamber models to directly visualize angiogenesis in relation to AZIN1 expression in real-time

What methods can detect subcellular redistribution of AZIN1 during different cell cycle phases?

Detecting the dynamic subcellular redistribution of AZIN1 during the cell cycle requires sophisticated imaging approaches:

• Live cell imaging techniques:

  • Generate stable cell lines expressing fluorescently-tagged AZIN1 (e.g., AZIN1-GFP)

  • Perform time-lapse confocal microscopy through complete cell cycles

  • Incorporate cell cycle phase markers (e.g., fluorescent PCNA for S-phase)

• Fixed cell analysis:

  • Synchronize cells and fix at specific cell cycle phases

  • Co-immunostain for AZIN1 and cell cycle markers:

    • Cyclin D1 (G1 phase)

    • PCNA (S phase)

    • Phospho-histone H3 (M phase)

  • Use high-content imaging systems for quantitative analysis

• Subcellular fractionation approaches:

  • Isolate nuclear, cytoplasmic, and centrosomal fractions from synchronized cells

  • Perform western blotting for AZIN1 in each fraction

  • Include purity controls (lamin B1 for nuclear, α-tubulin for cytoplasmic, γ-tubulin for centrosomal fractions)

• Quantification methods:

  • Calculate nuclear/cytoplasmic ratios using digital image analysis

  • Measure centrosomal AZIN1 intensity from prophase to late anaphase

  • Track colocalization coefficients between AZIN1 and OAZ1 throughout the cell cycle

• Advanced imaging approaches:

  • Super-resolution microscopy (STED, PALM, or STORM) for precise localization

  • FRET analysis to measure AZIN1-OAZ1 interaction dynamics in different cellular compartments during cell cycle progression

What novel applications are emerging for detecting AZIN1 RNA editing in clinical samples?

Emerging applications for detecting AZIN1 RNA editing in clinical contexts include:

• Antibody-based editing detection:

  • Development of conformation-specific antibodies that recognize the structural changes induced by S367G editing

  • Proximity ligation assays combining RNA probes with protein detection for simultaneous quantification of editing status and protein levels

• Clinical prognostic applications:

  • Multiplex IHC panels combining AZIN1 with other cancer biomarkers for improved patient stratification

  • Correlation of AZIN1 editing rates with treatment response and survival outcomes

  • Longitudinal monitoring of AZIN1 editing during cancer progression and treatment

• Liquid biopsy approaches:

  • Detection of edited AZIN1 in circulating tumor cells

  • Development of highly sensitive assays for edited AZIN1 protein in blood or other body fluids

• Integration with molecular diagnostics:

  • Correlation of AZIN1 RNA editing with other molecular alterations

  • Analysis of the 20% incidence rate of AZIN1 S367G editing observed in NSCLC within broader genomic contexts

  • Development of comprehensive biomarker panels including AZIN1 editing status

• Therapeutic monitoring applications:

  • Assessment of AZIN1 editing as a predictive biomarker for response to angiogenesis inhibitors

  • Monitoring of IL-8 pathway modulation in response to AZIN1-targeted interventions

  • Integration with immunotherapy response prediction

How can researchers design experiments to investigate AZIN1's role beyond polyamine regulation?

To investigate AZIN1's expanding roles beyond polyamine regulation, researchers should consider these experimental approaches:

• Protein-protein interaction screening:

  • Perform BioID or APEX proximity labeling with AZIN1 as the bait protein

  • Conduct yeast two-hybrid screens to identify novel interactors

  • Use protein arrays to detect direct binding partners

  • Validate interactions using reciprocal co-immunoprecipitation

• Transcriptional regulation investigation:

  • ChIP-seq to identify potential DNA binding sites of nuclear-localized edited AZIN1

  • RNA-seq comparing wild-type vs. edited AZIN1 expression to identify differentially expressed genes

  • Motif analysis to identify potential regulatory elements

• Pathway analysis approaches:

  • Phosphoproteomics to identify signaling pathways affected by AZIN1 editing

  • Metabolomics extending beyond polyamine metabolism

  • Systems biology integration of multiple omics datasets

• Structure-function studies:

  • Generate domain-specific AZIN1 mutants to map regions required for non-polyamine functions

  • Perform in silico structural modeling to predict conformational changes induced by editing

  • Use small molecule screening to identify compounds that selectively disrupt specific AZIN1 interactions

• Translational regulation investigation:

  • Ribosome profiling in models with altered AZIN1 status

  • Analysis of AZIN1's potential role in stress granule formation or mRNA processing

  • Investigation of AZIN1's influence on translation of specific mRNAs

This comprehensive framework for designing AZIN1 experiments will help researchers effectively explore its diverse roles beyond the well-established polyamine regulatory functions.