AGTRAP Antibody

What is AGTRAP and why is it significant in cancer research?

AGTRAP (Angiotensin II Receptor-Associated Protein) functions primarily by interacting directly with the Angiotensin II Type 1 Receptor (AT1R) to modulate its signaling . It appears to be a negative regulator of type-1 angiotensin II receptor-mediated signaling by regulating receptor internalization and mechanisms of receptor desensitization such as phosphorylation .

Research has revealed that AGTRAP plays a substantial role in tumor progression, with high expression significantly related to poor prognosis in multiple cancer types, including glioma, liver cancer, and kidney chromophobe . Pan-cancer analysis has shown that AGTRAP is highly expressed in breast cancer, lung adenocarcinoma, and several other cancers, making it an important target for oncology research .

How is AGTRAP involved in cellular signaling pathways relevant to disease?

AGTRAP is involved in several key signaling pathways:

• Immune-related pathways: Natural killer cell-mediated cytotoxicity, chemokine signaling, RIG-I-like receptor signaling, toll-like receptor signaling, and JAK-STAT signaling pathway

• Metabolism-related pathways: Taurine and hypotaurine metabolism, glycine, serine, and threonine metabolism

• Additional pathways: NF-κB and MAPK signaling pathways in hepatocellular carcinoma

Gene Set Enrichment Analysis (GSEA) has demonstrated that AGTRAP enrichment in these pathways may contribute to tumor development through immune modulation and metabolic reprogramming . The enrichment of chemokine signaling pathway was consistent with the abundant correlation between immunomodulators and AGTRAP, suggesting a function in regulating immunomodulators and promoting tumor progression .

What epitopes are commonly targeted in commercial AGTRAP antibodies?

Commercial AGTRAP antibodies target various regions of the protein, with several common epitopes:

The C-terminal region (particularly around amino acids 108-159) appears to be a frequently targeted epitope, suggesting this region may be particularly immunogenic or accessible for antibody binding .

How should researchers validate AGTRAP antibodies for experimental use?

Rigorous validation is essential before using AGTRAP antibodies in experiments:

• Western blot validation: Confirm the antibody detects a protein of the expected molecular weight. Note that when using recombinant proteins with tags (e.g., GST tag adds 26 KDa), the observed molecular weight may differ from the predicted value for native AGTRAP .

• Cross-reactivity testing: Some antibodies are validated against protein arrays containing hundreds of human recombinant protein fragments to ensure specificity. The Prestige Antibodies, for example, are tested on protein arrays of 364 human recombinant protein fragments .

• Tissue validation: Test the antibody on tissues known to express or lack AGTRAP. Commercial antibodies often undergo validation on tissue arrays of 44 normal human tissues and 20 common cancer types .

• Positive and negative controls: Include appropriate controls in each experiment, such as tissues with known AGTRAP expression patterns and secondary-only controls to assess background staining.

• Multiple antibody approach: When possible, use multiple antibodies targeting different epitopes to confirm findings, especially for novel observations.

What is the recommended protocol for AGTRAP immunohistochemistry?

Based on published methodologies for AGTRAP IHC staining:

• Sample preparation:

  • Collect tissue samples and fix in appropriate fixative

  • Embed in paraffin and section at 3mm thickness

  • Bake at 60°C for 2h for dewaxing

  • Rehydrate through graded alcohol series and wash with PBS

• Antigen retrieval and blocking:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 min at 25°C

  • Perform antigen retrieval by microwaving in 10 mM citrate buffer (pH 6.0) for 15 min

  • Block with 5% normal goat serum for 30 min at room temperature

• Antibody incubation:

  • Incubate with primary anti-AGTRAP antibody (typical dilutions range from 1:100 to 1:500) at 4°C overnight

  • Incubate with appropriate secondary antibody for 30 min at room temperature

• Detection and scoring:

  • Develop using streptavidin-peroxidase (SP) method according to kit instructions

  • Score based on intensity of immunostaining (0: colorless; 1: light-yellow; 2: brownish-yellow; 3: dark brown) multiplied by percentage of positive cells (0: 0%, 1: 1%-10%, 2: 11%-50%, 3: 51%-75%, 4: 76%-100%)

What are the key considerations for Western blot detection of AGTRAP?

For optimal Western blot results when detecting AGTRAP:

• Protein extraction:

  • Extract total protein using RIPA buffer

  • Centrifuge at 12,000×g for 15 min at 4°C to collect supernatant

  • Quantify protein concentration using BCA Protein Assay Kit

• Gel electrophoresis and transfer:

  • Separate equal amounts of protein by SDS-PAGE (12.5% gel recommended)

  • Transfer to PVDF membrane (0.22 μm)

• Antibody incubation:

  • Block with 5% skimmed milk in TBS for 1h

  • Incubate with primary anti-AGTRAP antibody (typical dilution 1:2000) at 4°C overnight

  • Incubate with secondary antibodies (typical dilution 1:2000) for 1h the next day

• Detection:

  • Visualize using enhanced chemiluminescence substrate

  • Quantify using an image analyzer system

How does AGTRAP expression correlate with immune infiltration in the tumor microenvironment?

AGTRAP expression shows significant correlation with immune cell infiltration across multiple cancer types:

• Positive correlation with infiltration of B cells (correlation = 0.225, p = 2.43e-05), CD8+ T cells (correlation = 0.321, p = 1.29e-09), CD4+ T cells (correlation = 0.292, p = 3.52e-08), macrophages (correlation = 0.417, p = 8.18e-16), neutrophils (correlation = 0.345, p = 4.22e-11), and dendritic cells (correlation = 0.406, p = 6.14e-15) in hepatocellular carcinoma .

• Association with immune scores: Significantly positive associations were found between AGTRAP and ImmuneScore in multiple cancers, including LGG (lower-grade glioma), NB (neuroblastoma), BLCA (bladder cancer), and several other cancer types .

• M2 macrophage correlation: AGTRAP expression was positively related to M2 macrophage infiltration among 25 cancer types . M2 macrophages promote angiogenesis, tissue reconstruction, and tumor progression .

How can bioinformatic approaches be used to study AGTRAP in cancer research?

Several bioinformatic approaches have been successfully employed to study AGTRAP:

• Expression analysis databases:

  • The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets for comparing expression between tumor and normal tissues

  • Cancer Cell Line Encyclopedia (CCLE) for analyzing expression across cancer cell lines

  • UCSC Xena Browser for standardized pan-cancer datasets

• Correlation analyses:

  • xCell algorithms using the "IOBR" R package to evaluate correlations between AGTRAP and immune scores and immune cell infiltration

  • Pearson's and Spearman's correlation analyses for assessing relationships between AGTRAP and other genes or immune parameters

• Survival analyses:

  • Kaplan-Meier plotter for assessing prognostic value

  • UALCAN for evaluating diagnostic value through ROC curves

• Pathway analyses:

  • Gene Set Enrichment Analysis (GSEA) to identify enriched pathways

  • Gene Ontology and KEGG pathway analyses using R software

What is known about AGTRAP's role in T-cell exhaustion and potential immunotherapy implications?

AGTRAP expression shows significant correlations with T-cell exhaustion markers and immunotherapy response:

• T-cell exhaustion markers: AGTRAP expression positively correlates with PD-1 (PDCD1), GZMB, LAG-3, CTLA-4, and HAVCR2 (TIM-3) in hepatocellular carcinoma, suggesting involvement in T-cell exhaustion pathways .

• Immunotherapy response prediction: Overexpression of AGTRAP predicts better immunotherapy responses in specific cancer types, including ACC (adrenocortical carcinoma), KICH (kidney chromophobe), GBM (glioblastoma), DLBC (diffuse large B-cell lymphoma), and COAD (colon adenocarcinoma) .

• Biomarker potential: The correlation with tumor mutation burden (TMB), microsatellite instability (MSI), and neoantigens suggests that AGTRAP could serve as a predictive biomarker for immunotherapy efficacy .

What is the prognostic significance of AGTRAP expression in different cancer types?

AGTRAP expression has significant prognostic implications across multiple cancer types:

How does AGTRAP expression differ between tumor and normal tissues across cancer types?

AGTRAP expression varies significantly between tumor and normal tissues across different cancer types:

These expression patterns have been confirmed through multiple methods, including bioinformatic analyses of TCGA data, immunohistochemistry, and Western blot validation on clinical specimens .

How might AGTRAP serve as a therapeutic target in cancer?

Based on current research findings, AGTRAP shows potential as a therapeutic target through several mechanisms:

• Modulation of immune responses: Given its correlation with immune cell infiltration and T-cell exhaustion markers, targeting AGTRAP might enhance anti-tumor immunity .

• Combination with immunotherapy: The correlation with immunotherapy response biomarkers suggests potential for combination approaches targeting AGTRAP alongside immune checkpoint inhibitors .

• Targeting associated signaling pathways: Interventions aimed at the NF-κB and MAPK signaling pathways that AGTRAP influences may provide therapeutic benefit in cancers where AGTRAP is overexpressed .

• RNA interference approaches: The validation of AGTRAP antibodies for RNAi applications suggests this as a potential approach for therapeutic development .

What are the key methodological challenges in studying AGTRAP in complex tissues?

Researchers face several challenges when studying AGTRAP in complex tissues:

• Cell-type specificity: AGTRAP is expressed in multiple cell types, including Kupffer cells and T cells in the liver, requiring careful interpretation of bulk tissue analysis .

• Subcellular localization: AGTRAP is primarily localized in cell plasma, which may require specialized techniques for accurate visualization and quantification .

• Post-translational modifications: Current data on AGTRAP post-translational modifications is limited, which may impact antibody recognition and functional studies .

• Isoform specificity: Multiple AGTRAP antibodies recognize different epitopes, potentially capturing different isoforms or modified forms of the protein, necessitating careful antibody selection .

• Cross-reactivity concerns: While most commercial antibodies are validated for human AGTRAP, cross-reactivity with mouse or rat orthologs varies, requiring validation for cross-species studies .