GBP4 Antibody

What is GBP4 and what are its primary functions in immunity?

GBP4 (Guanylate Binding Protein 4) is an interferon (IFN)-inducible GTPase that plays crucial roles in innate immunity against a diverse range of bacterial, viral, and protozoan pathogens . As a member of the GBP family, GBP4 belongs to the GB1/RHD3 GTPase family and efficiently hydrolyzes GTP to both GDP and GMP . GBP4 has dual immunological functions: it enhances immune cell infiltration while also potentially regulating T cell exhaustion mechanisms . In cancer biology, GBP4 has been identified as functionally relevant across numerous types of human cancers with context-dependent effects - displaying antitumor effects in some cancers (colorectal cancer, melanoma, breast cancer) while promoting tumor progression in others (pancreatic cancer, prostate cancer, ovarian cancer, and glioblastoma) .

Selection of the appropriate GBP4 antibody depends on multiple factors:

• Target species: Confirm reactivity with your species of interest (human, mouse, rat). For example, ABIN7444514 shows reactivity with mouse samples, while other antibodies like ab232693 are validated for human samples .

• Epitope recognition: Consider the specific region of GBP4 targeted by the antibody. Available antibodies target different amino acid regions:

  • AA 1-304 region (ABIN7444514)

  • AA 1-300 region (ab232693)

  • AA 1-350 region (ab232689)

  • C-terminal regions (various antibodies)

• Application compatibility: Verify that the antibody has been validated for your specific application with recommended dilutions. For Western blot applications, typical dilutions range from 1:500-1:3000 depending on the antibody .

• Validation data: Review immunoblots, IHC images, and citation records to assess antibody performance and specificity in contexts similar to your experimental design .

What are the recommended protocols for using GBP4 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of GBP4 in tissue samples:

• Sample preparation: Use standard formalin-fixed, paraffin-embedded (FFPE) tissue sections or tissue microarrays (TMAs) .

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) .

• Blocking: Block endogenous peroxidase activity with hydrogen peroxide and prevent non-specific binding with appropriate serum or protein block .

• Primary antibody incubation: For GBP4 detection, optimize concentration based on the specific antibody:

  • anti-human GBP4 (Ab232693, Abcam): 1:2000 dilution

  • ab232689: 20 μg/ml for mouse tissues

• Detection system: Use appropriate HRP-conjugated secondary antibodies followed by DAB visualization and hematoxylin counterstaining .

• Scoring method: Score GBP4 expression according to staining intensity and percentage of positive cells: 0–5% (0), 5–25% (1), 25–50% (2), 50–75% (3), and 75–100% (4). Expression levels can be classified as negative (0–3, −), weakly positive (4, +), moderately positive (6, ++), or strongly positive (> 6, +++) .

What controls should I include when working with GBP4 antibodies?

For rigorous validation of GBP4 antibody experiments:

• Positive controls: Use tissues/cells known to express GBP4:

  • Human: HeLa cells, A431 cells, placenta tissue

  • Mouse: Heart tissue, testis, kidney, stomach

• Negative controls: Include:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Tissues known to have low GBP4 expression

• Knockdown/knockout validation: For definitive specificity validation, include samples with GBP4 gene silencing using shRNA constructs (e.g., pLVX-GBP4-shRNA-GFP) .

• Recombinant protein: Use recombinant GBP4 protein as a positive control in Western blot applications .

How can I optimize Western blot protocols for GBP4 detection?

For successful GBP4 detection in Western blot applications:

• Sample preparation: Extract total protein using RIPA buffer supplemented with protease inhibitors.

• Protein loading: Load 20-30 μg of total protein per lane.

• Gel selection: Use 10% SDS-PAGE gels to resolve GBP4 protein (predicted MW: 73 kDa) .

• Transfer conditions: Transfer proteins to PVDF membranes (0.45 μm) using standard wet transfer conditions.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody dilution: Use optimized dilutions for primary antibody incubation:

  • Proteintech 17746-1-AP: 1:500-1:3000

  • ab232689: 1 μg/mL

• Detection: Use HRP-conjugated secondary antibodies (typically 1:2000-1:5000 dilution) and ECL detection reagents .

• Predicted band size: Expect to detect GBP4 at approximately 73 kDa .

How can GBP4 be used as a biomarker in cancer immunotherapy research?

GBP4 shows significant potential as a biomarker for cancer immunotherapy research:

• Predictive value for immunotherapy response: GBP4 expression levels have been correlated with response to anti-PD-1 therapy in several studies. In GSE135222 dataset, GBP4 expression was significantly higher in complete response (CR) groups compared to non-responder (NR) groups .

• Correlation with T-cell-inflamed phenotype: GBP4 expression positively correlates with T-cell-inflamed gene expression profile (GEP) scores, which serve as surrogate measures for predicting clinical responses to anti-PD-1 therapy .

• Association with immune checkpoint expression: GBP4 shows strong positive correlations with immune checkpoint genes (PDCD1, CD274, TIGIT, CTLA4) across multiple cancer types, particularly in non-small cell lung cancer (NSCLC) .

• Tumor microenvironment characterization: High GBP4 expression is associated with:

  • Increased immune cell infiltration

  • Higher immune and stromal scores

  • Lower tumor purity

  • Enhanced expression of chemokines and immunomodulators

Researchers should consider integrating GBP4 expression analysis into biomarker panels for patient stratification in immunotherapy clinical trials .

What methods can be used to study the relationship between GBP4 and T cell exhaustion?

To investigate GBP4's role in T cell exhaustion, researchers can employ several methodological approaches:

• Multiplex immunohistochemistry: Use fluorescent multiplex IHC to simultaneously assess GBP4 expression along with T cell exhaustion markers:

  • Count TCF7+, PD-1+, TCF1+PD-1+ TILs, and TCF1−PD-1+ TILs from high-power fields (HPFs)

  • Analyze co-localization patterns of GBP4 with T cell exhaustion markers

• Flow cytometry: Analyze T cell exhaustion phenotypes in relation to GBP4 levels:

  • Assess expression of multiple checkpoint receptors (PD-1, TIGIT, LAG-3, TIM-3)

  • Evaluate T cell functional markers (IFN-γ, TNF-α, IL-2 production)

  • Measure proliferation capacity and cytotoxic function

• In vitro T cell killing assays: Test how GBP4 expression affects T cell-mediated cytotoxicity:

  • Use primary organoids with varying GBP4 expression levels

  • Assess sensitivity to anti-PD-1 treatment in relation to GBP4 levels

• Chemotaxis assays: Evaluate how GBP4 affects T cell recruitment and infiltration .

• Gene expression profiling: Analyze correlation between GBP4 and exhaustion-related gene signatures in patient samples.

What is known about the epigenetic regulation of GBP4 and how can this be experimentally validated?

GBP4 expression is regulated through epigenetic mechanisms, particularly DNA methylation. Researchers can investigate this relationship using:

• Methylation analysis techniques:

  • Bisulfite genomic sequencing to assess methylation status of GBP4 regulatory regions

  • Analysis using the Shiny Methylation Analysis Resource Tool (SMART) to evaluate DNA methylation levels in The Cancer Genome Atlas (TCGA) samples

• Targeted methylation approaches:

  • dCas9-SunTag-DNMAT3A-sgRNA-targeted methylation system for selective methylation of specific DNA loci to validate regulatory roles on GBP4 expression

  • Design sgRNAs targeting regulatory regions of GBP4 gene

• Methylation inhibitor studies:

  • Treat cells with DNA methyltransferase inhibitors (e.g., 5-azacytidine) to observe effects on GBP4 expression

  • Perform dose-response and time-course experiments to characterize methylation-dependent regulation

• Correlation analyses:

  • Compare GBP4 expression patterns with DNA methylation levels across different cancer types

  • Identify CpG sites with significant negative correlation to expression

Research has demonstrated that DNA hypo-methylation in regulatory regions of GBP4 in pancreatic ductal adenocarcinoma influences its expression levels, suggesting a potential therapeutic target for epigenetic modulation .

How can researchers evaluate GBP4's role in shaping the tumor microenvironment?

To comprehensively assess GBP4's impact on the tumor microenvironment (TME):

• TME component analysis:

  • Use ESTIMATE method to evaluate tumor purity, immune score, and stromal score in relation to GBP4 expression

  • Apply multiple independent algorithms (CIBERSORTx, etc.) to assess immune cell infiltration patterns

• Chemokine/cytokine profiling:

  • Analyze expression of chemokines (CXCL9, CXCL10, CXCL11) in relation to GBP4 levels

  • Perform knockdown/overexpression studies of GBP4 and measure effects on chemokine production

• Signaling pathway analysis:

  • Investigate STAT1/NK axis involvement in GBP4-mediated immune infiltration

  • Assess IFN-γ/STAT1 and TNF-α/NF-κB signaling in relation to GBP4 expression

• In vivo modeling:

  • Generate GBP4-overexpressing or knockout tumor models

  • Analyze tumor growth kinetics and functional status of tumor-infiltrating CD8+ T cells

  • Evaluate response to immunotherapy in these models

• Spatial transcriptomics/proteomics:

  • Map GBP4 expression patterns in relation to different TME zones (tumor nest, invasive margin, tertiary lymphoid structures)

  • Correlate with spatial distribution of immune cells and functional markers

Research indicates that high GBP4 expression is associated with an inflamed TME characterized by increased immune cell infiltration, higher expression of immune checkpoint genes, and potentially enhanced response to immunotherapy .

What are common issues with GBP4 antibody specificity and how can they be addressed?

Researchers may encounter several specificity challenges when working with GBP4 antibodies:

• Cross-reactivity with other GBP family members: The human GBP family includes seven members (GBP1-7) with structural similarities . To address this:

  • Select antibodies raised against unique epitopes of GBP4

  • Validate with recombinant GBP4 protein and compare with other recombinant GBP proteins

  • Perform siRNA/shRNA knockdown of GBP4 to confirm specificity

• Species-specific differences: Human GBP4 shares varying degrees of sequence homology with mouse (48%) and rat (49%) orthologs , which may affect cross-species reactivity. To address this:

  • Use species-specific positive controls to validate antibody performance

  • Select antibodies validated for your species of interest

  • Consider the homology of the immunogen sequence across species

• Background staining in IHC applications: To minimize:

  • Optimize antigen retrieval conditions

  • Adjust antibody concentrations (start with manufacturer's recommendations)

  • Include robust blocking steps to prevent non-specific binding

  • Use IgG isotype controls at equivalent concentrations

• Multiple bands in Western blot: May indicate degradation products, post-translational modifications, or non-specific binding. To address:

  • Use fresh samples with protease inhibitors

  • Optimize sample preparation conditions

  • Run additional controls (recombinant protein, knockdown samples)

How can I verify GBP4 antibody performance in my experimental system?

To ensure reliable GBP4 detection in your specific experimental setup:

• Multi-antibody validation approach:

  • Use at least two different GBP4 antibodies targeting different epitopes

  • Compare staining/binding patterns between antibodies

  • Prioritize antibodies with published validation records

• Expression induction validation:

  • Treat cells with interferon to upregulate GBP4 expression (GBP4 is interferon-inducible)

  • Compare expression levels between treated and untreated samples

  • Confirm upregulation at both protein and mRNA levels

• Genetic manipulation controls:

  • Perform shRNA knockdown using validated constructs (e.g., pLVX-GBP4-shRNA-GFP)

  • Create GBP4 overexpression systems (e.g., pLVX-GBP4-GFP)

  • Use these genetic controls in parallel with your experimental samples

• Correlation with orthogonal techniques:

  • Compare protein detection (Western blot/IHC) with mRNA expression (qRT-PCR)

  • Confirm subcellular localization patterns using fluorescence microscopy

• Titration experiments:

  • Test multiple antibody dilutions to determine optimal signal-to-noise ratio

  • Document linearity of detection within relevant concentration ranges

How should contradictory results regarding GBP4 function be interpreted and reconciled?

When confronted with contradictory findings regarding GBP4 function:

• Context-dependent effects: GBP4 appears to have distinct roles in different cancer types - antitumor effects in colorectal cancer, melanoma, and breast cancer, but tumor-promoting effects in pancreatic, prostate, ovarian cancers, and glioblastoma . Consider:

  • Tissue-specific microenvironmental factors

  • Different genetic backgrounds between experimental models

  • Varying immune contextures between cancer types

• Dual immunological roles: GBP4 can simultaneously enhance T cell infiltration while inducing T cell exhaustion . This apparent contradiction may reflect:

  • Temporal dynamics of immune response

  • Compensatory immune regulatory mechanisms

  • Balance between pro-inflammatory and regulatory signals

• Methodological differences: Discrepancies may arise from:

  • In vitro vs. in vivo studies

  • Different model systems (cell lines, primary cultures, animal models)

  • Varying experimental conditions (cytokine stimulation, timing, etc.)

• Threshold effects: Consider whether GBP4 functions may depend on expression levels, with different effects at low versus high expression.

• Integration approach: To reconcile contradictory findings:

  • Implement multiple complementary experimental approaches

  • Consider temporal dynamics in your experimental design

  • Validate findings across different model systems

  • Integrate in vitro mechanisms with in vivo phenotypes

Research suggests GBP4's complex role in tumor immunity may make it a potentially valuable biomarker for predicting immunotherapy response while its context-dependent effects require careful experimental design to properly characterize .

How might GBP4 be leveraged for predicting response to combination immunotherapies?

Recent research suggests GBP4 may have utility in predicting response to combination immunotherapies:

• Dual marker potential: GBP4 expression correlates with both T cell infiltration and exhaustion markers, making it potentially valuable for identifying patients who may benefit from combination approaches targeting both trafficking and exhaustion mechanisms .

• Predictive modeling: Research could focus on:

  • Integrating GBP4 expression with other biomarkers in predictive algorithms

  • Creating GBP4-based expression signatures with machine learning approaches

  • Developing thresholds for stratifying patients into treatment groups

• Combination therapy applications:

  • Anti-PD-1 plus anti-CTLA-4: GBP4 high tumors showed significantly higher sensitivity to anti-PD-1 treatment in primary organoid models

  • Epigenetic modifiers plus immunotherapy: Given GBP4's regulation by DNA methylation, combinations of DNA methyltransferase inhibitors with immune checkpoint inhibitors could be investigated

  • Anti-EGFR therapy plus immunotherapy: GBP4 expression may predict enhanced responsiveness to anti-EGFR therapy in addition to immunotherapy

• Mechanistic studies needed:

  • Elucidate how GBP4 simultaneously affects T cell recruitment and exhaustion

  • Determine how GBP4-mediated effects on the tumor microenvironment influence treatment response

  • Investigate whether GBP4 expression changes during treatment could serve as pharmacodynamic biomarkers

What methodological advances are needed to better characterize GBP4's role in immune cell function?

Several methodological innovations could advance understanding of GBP4's immunological functions:

• Single-cell approaches:

  • Single-cell RNA sequencing to characterize GBP4 expression patterns across immune cell subtypes

  • Single-cell proteomics to correlate GBP4 protein levels with functional immune cell states

  • Spatial single-cell analysis to map GBP4+ cells within the tumor microenvironment

• Live-cell imaging techniques:

  • Develop fluorescent reporter systems for real-time tracking of GBP4 expression

  • Use intravital microscopy to visualize GBP4-expressing cells in vivo

  • Track dynamic changes in GBP4 expression during immune cell activation/exhaustion

• Functional immune assays:

  • Advanced T cell killing assays with real-time monitoring capabilities

  • High-dimensional flow cytometry panels to correlate GBP4 with multiple exhaustion markers

  • Organoid co-culture systems with immune components to model GBP4-mediated effects

• CRISPR-based approaches:

  • CRISPR activation/inhibition systems to modulate GBP4 expression with temporal control

  • CRISPR screens to identify GBP4 regulators and downstream effectors

  • Base editing approaches to introduce specific mutations in GBP4 regulatory regions

• Improved animal models:

  • Generate conditional GBP4 knockout models in specific immune cell populations

  • Develop humanized mouse models expressing human GBP4 for more translationally relevant studies

  • Create reporter mice for in vivo tracking of GBP4 expression