ANXA9 Antibody

What is the molecular structure and function of ANXA9?

ANXA9 is a unique member of the annexin family of calcium-dependent phospholipid-binding proteins. Unlike typical annexins, ANXA9 contains four homologous type II calcium-binding sites in its conserved tetrad core with amino acid substitutions that alter their function. Despite these modifications, structural analysis indicates the putative ion channel formed by the tetrad core remains intact . Functionally, ANXA9 serves as a low-affinity receptor for acetylcholine and is targeted by pemphigus vulgaris antibodies in keratinocytes . The protein has a calculated and observed molecular weight of 38 kDa and consists of 345 amino acids encoded by the BC005830 gene sequence .

What are the recommended applications and dilutions for ANXA9 antibodies?

ANXA9 antibodies have been validated for multiple experimental applications with specific optimal conditions:

These dilutions should be optimized for each specific experimental system as results may be sample-dependent .

How do I validate ANXA9 antibody specificity in my experimental system?

Proper validation of ANXA9 antibody specificity requires a multi-faceted approach:

• Positive control testing: Verify antibody reactivity using known ANXA9-expressing samples such as A431 cells, MCF-7 cells, human fetal kidney lysates, or human tonsil lysates for Western blot applications .

• Size verification: Confirm that the detected band aligns with the expected molecular weight of 38 kDa .

• siRNA knockdown validation: Implement ANXA9-specific siRNA knockdown as a negative control. Successful knockdown should significantly reduce the antibody signal, confirming specificity. Recommended siRNA sequences include:

  • si-ANXA9-1: 5′-GGCAGCUCAUCUCACGAAATT-3′

  • si-ANXA9-2: 5′-GGACGUGGCCAUUGAAAUUTT-3′

  • si-ANXA9-3: 5′-GCAGUCUACAAACACAAUUTT-3′

• Immunoprecipitation confirmation: Perform immunoprecipitation followed by Western blot detection to verify antibody specificity through enrichment of the target protein .

• Tissue-specific expression validation: Compare antibody reactivity across multiple tissue types known to express ANXA9, including human breast cancer, colon cancer, heart, kidney, liver, placenta, skin, spleen, and testis tissues .

What is the optimal protocol for qRT-PCR analysis of ANXA9 expression?

For accurate ANXA9 expression analysis via qRT-PCR, the following standardized protocol is recommended:

Primer design:

• Forward primer: 5′-TGAGCCCAATTACCAAGTCC-3′ (located in exon 13)

• Reverse primer: 5′-GTTCAGCCAAACACGGAAAT-3′ (located in exon 14)

Reference gene primers (GAPDH):

• Forward: 5′-TTGGTATCGTGGAAGGACTCA-3′

• Reverse: 5′-TGTCATCATATTGGCAGGTT-3′

PCR conditions:

• 35 cycles with parameters: 95°C for 40 sec, 45°C for 40 sec, 72°C for 60 sec

• For quantitative assessment, use a real-time PCR system with appropriate master mix (e.g., LightCycler FastStart DNA Master SYBR Green I kit)

Data analysis:

• Use the 2^(-ΔΔCt) method for calculating differences in gene expression levels

• Normalize ANXA9 expression to GAPDH expression for each sample

• For tumor studies, calculate the ratio of ANXA9 expression in tumor tissue to that in adjacent normal tissue

How do I optimize immunohistochemistry protocols for ANXA9 detection?

Successful ANXA9 immunohistochemistry requires careful optimization of multiple parameters:

Sample preparation:

• Fix tissues in appropriate fixative (typically 4% paraformaldehyde)

• Process and embed in paraffin following standard protocols

• Section at 4-5 μm thickness

Antigen retrieval options:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Antibody incubation:

• Primary antibody dilution: 1:50-1:500 (start with 1:100 and optimize)

• Incubation time: Typically overnight at 4°C or 1-2 hours at room temperature

• Secondary antibody: Select based on primary antibody host species (typically rabbit IgG for ANXA9)

Detection systems:

• DAB (3,3'-diaminobenzidine) for brightfield microscopy

• Fluorophore-conjugated secondary antibodies for fluorescence applications

Positive control tissues:
Include one or more of the following validated positive control tissues: human breast cancer, colon cancer, heart, kidney, liver, placenta, skin, spleen, or testis tissue .

How does ANXA9 expression correlate with cancer prognosis across different malignancies?

ANXA9 has emerged as a significant prognostic biomarker across multiple cancer types, with consistent patterns of correlation between expression levels and patient outcomes:

Colorectal cancer (CRC):

Gastric cancer (GC):

• ANXA9 serves as a prognostic indicator related to immune responses

• Expression patterns correlate with clinicopathological parameters including survival time

Breast cancer:

• High ANXA9 expression found predominantly in metastatic breast cancer tissues

• Expression levels correlate directly with disease progression

• Mechanistically linked to tumor growth and lung metastasis in xenograft models

These findings consistently demonstrate that ANXA9 overexpression is associated with poorer clinical outcomes across multiple cancer types, suggesting its potential utility as a universal cancer prognostic marker.

What signaling pathways does ANXA9 regulate in cancer progression?

Research has identified several key signaling pathways through which ANXA9 influences cancer development and progression:

Wnt signaling pathway:

• ANXA9 promotes colorectal cancer progression by positively regulating Wnt signaling

• Knockdown of ANXA9 inhibits CRC cell proliferation through Wnt pathway modulation

AKT/mTOR/STAT3 pathway:

• In breast cancer, ANXA9 mediates S100A4 signaling to regulate the AKT/mTOR/STAT3 pathway

• This regulation impacts p53/Bcl-2-mediated apoptosis

• ANXA9 phosphorylation at Ser2 and Thr69 sites appears critical for these effects

Immune-related pathways:

• Low ANXA9 expression associates with activation of immune pathways including:

  • Chemokine binding

  • Leukocyte adhesion to vascular endothelial cells

  • Positive regulation of leukocyte cell-cell adhesion

  • T cell selection

  • Response to chemokine signaling

Cytokine regulation:

• ANXA9 mediates the excretion of pro-tumorigenic cytokines including IL-6, IL-8, CCL2, and CCL5

• These cytokines contribute to angiogenesis and tumor microenvironment modulation

Understanding these pathway interactions provides potential targets for therapeutic intervention in ANXA9-expressing cancers.

How can I effectively knockdown ANXA9 expression for functional studies?

For efficient ANXA9 knockdown in experimental systems, the following comprehensive approach is recommended:

siRNA transfection protocol:

• Cell preparation: Seed HT29, HCT116 (CRC) or other appropriate cancer cell lines at 60-70% confluence

• Transfection mix preparation:

  • Dilute siRNA in OPTI-MEM (Invitrogen)

  • Prepare Lipofectamine 2000 (Invitrogen) in OPTI-MEM

  • Combine and incubate for 20 minutes at room temperature

• Transfection: Add transfection mix to cells and incubate for 48-72 hours

Validated siRNA sequences:

• si-ANXA9-1: 5′-GGCAGCUCAUCUCACGAAATT-3′

• si-ANXA9-2: 5′-GGACGUGGCCAUUGAAAUUTT-3′

• si-ANXA9-3: 5′-GCAGUCUACAAACACAAUUTT-3′

Knockdown validation methods:

• RNA level: qRT-PCR using specific ANXA9 primers (see section 2.1)

• Protein level: Western blot using validated ANXA9 antibodies at 1:500-1:2000 dilution

Functional assays following knockdown:

• Proliferation: EdU assay with fluorescence microscopy detection

• Colony formation: 1000 cells/well in 6-well plates, fixed after 14 days, stained with crystal violet

• Invasion/migration: Transwell or wound healing assays

• Apoptosis: Flow cytometry with Annexin V/PI staining

Additionally, in vivo models using ANXA9-knockdown cells in xenograft experiments can validate the effects on tumor growth and metastasis .

How can I investigate ANXA9's role in immune cell infiltration and tumor microenvironment?

ANXA9 has emerged as a potential regulator of tumor immune microenvironment. To investigate this relationship, implement this comprehensive strategy:

Bioinformatic approaches:

• Divide samples into ANXA9-high and ANXA9-low expression groups based on median or quartile expression levels

• Utilize the Tumor Immune Estimation Resource (TIMER) to analyze correlations between ANXA9 expression and immune cell infiltration

• Perform single-sample Gene Set Enrichment Analysis (ssGSEA) to quantify immune cell populations in each sample

• Focus analysis on the following 21 immune-related gene sets that show significant differences between ANXA9 expression groups:

  • Antigen presentation: APC_co_inhibition, DCs, pDCs, HLA

  • T cell populations: CD8+_T_cells, T_helper_cells, Tfh, Th1_cells, Th2_cells, Treg, TIL

  • T cell regulation: T_cell_co-inhibition, T_cell_co-stimulation, Check.point

  • Other immune components: B_cells, CCR, Cytolytic_activity, Inflammation.promoting, Neutrophils, NK_cells, Type_II_IFN_Response

Experimental validation approaches:

• Immunohistochemistry:

  • Perform multiplexed IHC to simultaneously detect ANXA9 and immune cell markers

  • Analyze spatial relationships between ANXA9-expressing cells and immune infiltrates

• Flow cytometry:

  • Isolate cells from ANXA9-high and ANXA9-low tumors

  • Quantify immune cell populations using appropriate markers

  • Compare immune profile differences between expression groups

• Cytokine profiling:

  • Measure levels of key cytokines (IL-6, IL-8, CCL2, CCL5) in ANXA9-high versus ANXA9-low samples

  • Correlate cytokine levels with immune cell infiltration patterns

This integrated approach enables comprehensive characterization of ANXA9's influence on tumor immune microenvironment.

What experimental design is recommended to study ANXA9's role in cancer metastasis?

To thoroughly investigate ANXA9's contribution to metastatic processes, implement this multi-level experimental framework:

In vitro metastasis-related assays:

• Migration assays:

  • Wound healing (scratch) assay comparing ANXA9-knockdown vs. control cells

  • Transwell migration assay to quantify directional cell movement

• Invasion assays:

  • Matrigel-coated transwell assays to assess invasive capacity

  • 3D spheroid invasion assays in extracellular matrix

• Epithelial-mesenchymal transition (EMT) analysis:

  • Assess expression of EMT markers (E-cadherin, N-cadherin, vimentin) by Western blot and immunofluorescence

  • Compare EMT marker expression between ANXA9-knockdown and control cells

In vivo metastasis models:

• Lung metastasis model:

  • Inject ANXA9-knockdown or control cancer cells via tail vein

  • Quantify lung metastatic nodules

  • Histological and immunohistochemical analysis of metastatic lesions

• Spontaneous metastasis model:

  • Establish primary tumors using ANXA9-knockdown or control cells

  • Monitor primary tumor growth and spontaneous metastasis formation

  • Analyze both primary and metastatic lesions for ANXA9 expression and pathway activation

Molecular mechanism investigation:

• S100A4 interaction:

  • Analyze S100A4 levels in ANXA9-knockdown vs. control cells

  • Perform co-immunoprecipitation to confirm ANXA9-S100A4 interaction

  • Conduct rescue experiments with S100A4 overexpression in ANXA9-knockdown cells

• Phosphorylation analysis:

  • Generate phospho-deficient mutants at Ser2 and Thr69 sites of ANXA9

  • Compare metastatic potential of wild-type vs. phospho-mutant ANXA9

• Cytokine secretion profile:

  • Quantify IL-6, IL-8, CCL2, and CCL5 levels in conditioned media from ANXA9-modified cells

  • Evaluate impact of these cytokines on endothelial cell migration and angiogenesis

This comprehensive approach enables detailed characterization of ANXA9's role in cancer metastasis across multiple experimental systems.

How do I resolve common issues with ANXA9 Western blot detection?

Researchers frequently encounter challenges when detecting ANXA9 via Western blot. The following troubleshooting guide addresses common issues:

High background signal:

• Increase blocking time (try 1-2 hours at room temperature or overnight at 4°C)

• Use 5% BSA in TBST instead of milk for blocking and antibody dilution

• Increase washing time and volume (minimum 3×10 minutes with TBST)

• Dilute primary antibody further (try 1:2000 instead of 1:500)

• Ensure secondary antibody is highly cross-adsorbed to prevent non-specific binding

Weak or absent signal:

• Increase protein loading (start with 30-50 μg total protein)

• Decrease primary antibody dilution (try 1:500 instead of 1:2000)

• Extend primary antibody incubation time (overnight at 4°C)

• Use enhanced chemiluminescence detection system with longer exposure times

• Verify expression in your sample type (use A431 or MCF-7 cells as positive controls)

Multiple bands or incorrect molecular weight:

• Ensure complete protein denaturation (heat samples at 95°C for 5 minutes)

• Optimize gel percentage (10-12% acrylamide gels work well for 38 kDa ANXA9)

• Verify sample integrity (avoid repeated freeze-thaw cycles)

• Use freshly prepared lysates with protease inhibitors

• Consider that post-translational modifications may cause slight shifts in molecular weight

Inconsistent loading control:

• Verify equal loading using total protein stains (Ponceau S or SYPRO Ruby)

• Select appropriate housekeeping protein controls (GAPDH works consistently with ANXA9)

• Strip and reprobe membrane carefully if detecting multiple proteins

What factors affect ANXA9 antibody performance in immunohistochemistry applications?

Successful ANXA9 immunohistochemistry depends on careful optimization of multiple parameters:

Fixation considerations:

• Overfixation can mask epitopes; limit fixation time to 24 hours

• Underfixation leads to poor morphology and inconsistent staining

• Consider testing multiple fixatives if experiencing difficulties

Critical antigen retrieval factors:

• Buffer pH significantly impacts ANXA9 detection (pH 9.0 TE buffer is recommended primary method)

• Retrieval time and temperature affect epitope accessibility (try 20 minutes at 95-98°C)

• Cooling time after retrieval influences antibody binding (allow 20-30 minutes cooling)

Antibody dilution optimization:

• Start with manufacturer's recommended range (1:50-1:500)

• Prepare dilution series to determine optimal concentration for your specific tissue

• Consider tissue-specific optimization as different tissues may require different dilutions

Detection system selection:

• Polymer-based detection systems often provide better signal-to-noise ratio for ANXA9

• Biotin-based systems may give higher background in tissues with endogenous biotin

• Automated staining platforms can improve consistency but require specific protocol optimization

Common tissue-specific challenges:

• Tissues with high endogenous peroxidase activity require thorough quenching

• Tissues with high background may benefit from additional blocking steps

• Cancer tissues with heterogeneous expression require careful interpretation