GIPC2 Antibody

What is GIPC2 and why is it important in cancer research?

GIPC2 encodes a 315-amino acid protein that is 62% identical to GIPC1 and is considered a paralog of GIPC1 that can promote proliferation and invasion in tumor metastasis. The significance of GIPC2 varies across different cancer types - it shows increased expression in metastatic prostate cancer and appears to promote cancer cell invasion and migration . Interestingly, in colon adenocarcinoma (COAD), high GIPC2 expression is associated with favorable prognosis and increased infiltration of immune cells . These contrasting roles make GIPC2 an important target for comprehensive cancer research.

What methods are most effective for detecting GIPC2 expression in tissue samples?

Immunohistochemistry (IHC) with horseradish peroxidase is a reliable method for detecting GIPC2 protein in tissue samples. For optimal results, use a concentrated rabbit polyclonal antibody against human GIPC2 protein (such as BIOSS cat no. KT22301, 1:200 dilution) . Expression can be quantified using a scoring system that combines staining intensity and percentage of positive cells. Additionally, RT-PCR and western blotting are effective for analyzing GIPC2 mRNA and protein expression levels in cell lines and fresh tissue samples .

How should GIPC2 antibody specificity be validated?

Proper validation of GIPC2 antibodies should include positive and negative controls. Normal adult kidney tissue can be used as a positive control, while PBS can serve as a negative control . Western blot analysis should show a band at approximately 35 kDa (the expected molecular weight of GIPC2). Cross-reactivity with GIPC1 should be evaluated due to their 62% sequence identity . For further validation, testing the antibody on samples where GIPC2 has been knocked down via siRNA can confirm specificity.

How do epigenetic modifications influence GIPC2 expression, and how can this be studied with antibodies?

GIPC2 expression is regulated by promoter methylation, with distinct patterns observed across cancer types. In prostate cancer, promoter demethylation appears to activate GIPC2 expression . Researchers can investigate this relationship by combining GIPC2 antibody-based detection methods (IHC, western blot) with methylation analysis techniques such as the EpiTYPER MassARRAY System for quantitative DNA-methylation analysis . Treatment of cell lines with DNMT1 inhibitors like DAC (5-aza-2'-deoxycytidine) followed by GIPC2 antibody detection can demonstrate the direct relationship between demethylation and protein expression. This approach revealed that RWPE-1, C4-2, and Du145 cell lines demonstrated gradual demethylation and significant increases in GIPC2 mRNA and protein expression after DAC treatment .

What are the challenges in interpreting contradictory GIPC2 expression patterns across different cancer types?

One of the greatest challenges in GIPC2 research is reconciling its apparently contradictory roles across cancer types. In prostate cancer, GIPC2 acts as an oncogene promoting metastasis , while in colon adenocarcinoma, high GIPC2 expression is associated with favorable prognosis . When using GIPC2 antibodies for cross-cancer comparisons, researchers must consider tissue-specific microenvironments, genetic backgrounds, and signaling pathway contexts. Antibody-based tissue microarray studies across multiple cancer types, combined with pathway analysis tools such as Gene Set Enrichment Analysis (GSEA), can help elucidate these tissue-specific functions. In COAD, GIPC2 expression is enriched in cell cycle checkpoints, DNA replication, and mitosis-associated signaling pathways , while in prostate cancer, GIPC2 affects cellular functions related to cell movement and molecular binding .

How can GIPC2 antibodies be optimized for multiplexed imaging with other cancer biomarkers?

For advanced spatial biology studies, optimizing GIPC2 antibodies for multiplexed imaging requires careful selection of compatible antibodies that don't cross-react and appropriate fluorophore combinations to minimize spectral overlap. When studying prostate cancer metastasis, combine GIPC2 antibodies with markers for Wnt signaling components (particularly Fzd7) , as GIPC2 impacts PCa metastasis through Wnt signaling. For COAD research, multiplexing with immune checkpoint markers (CD274, CTLA4, HAVCR2, LAG3, PDCD1, PDCD1LG2, TIGIT, and SIGLEC15) is valuable as these genes show differential expression between high and low GIPC2 expression groups . Sequential staining protocols or tyramide signal amplification may be necessary to achieve clear signal separation in highly multiplexed panels.

What animal models are most appropriate for studying GIPC2 function using antibody-based detection methods?

For in vivo studies of GIPC2 function, particularly in metastasis research, cardiac injection models in male athymic nude mice have proven effective. Researchers have successfully established C4-2 and DU145 cell lines with stably downregulated GIPC2 and implanted them into the left cardiac ventricle of nude mice . GIPC2 antibody-based detection can be used for ex vivo imaging and pathological confirmation of metastasis sites, particularly in organs like the liver where marked metastasis differences were observed between control and GIPC2-knockdown groups . When designing these experiments, consider using bioluminescence imaging to monitor metastatic growth followed by antibody-based validation of GIPC2 expression in harvested tissues.

What are the critical factors to consider when selecting a GIPC2 antibody for specific research applications?

When selecting a GIPC2 antibody, researchers should consider: (1) Epitope specificity - choose antibodies targeting unique regions of GIPC2 to avoid cross-reactivity with GIPC1; (2) Validation status - prefer antibodies validated in multiple applications (IHC, western blot, immunofluorescence) with published research supporting their specificity; (3) Host species - consider the experimental design and secondary detection methods; (4) Clonality - monoclonal antibodies offer higher reproducibility while polyclonal antibodies may provide stronger signals; (5) Application compatibility - ensure the antibody works in your specific application (fixed tissues vs. frozen sections, denatured vs. native protein detection). For IHC applications in colon tissue, rabbit polyclonal antibodies at 1:200 dilution have demonstrated good results .

How can researchers optimize immunohistochemistry protocols for GIPC2 detection in different tissue types?

Optimizing IHC protocols for GIPC2 detection requires consideration of tissue-specific characteristics. For prostate tissues, positive staining is predominantly observed in metastatic neoplasms from bones, requiring potential decalcification considerations . For colon tissues, standardized scoring systems combining staining intensity (0-3) and percentage of positive cells (0-4) can be employed, with scores >3 classified as positive . General optimization considerations include: (1) Antigen retrieval method and duration; (2) Antibody concentration and incubation time; (3) Detection system sensitivity; (4) Counterstaining intensity to provide adequate contrast; (5) Inclusion of appropriate positive controls (kidney tissue) and negative controls (PBS) . Automated staining platforms can improve reproducibility for multi-sample studies.

What methods can be used to quantitatively analyze GIPC2 expression in cell and tissue samples?

Several quantitative methods can be employed for GIPC2 expression analysis:

• RT-PCR for mRNA quantification: This technique allows precise measurement of GIPC2 mRNA levels, as demonstrated in studies comparing expression between normal prostate cell lines and metastatic prostate cancer cell lines .

• Western blot with densitometry: For protein level quantification, western blotting followed by densitometric analysis can determine relative GIPC2 protein expression levels across different samples .

• IHC scoring systems: Semi-quantitative evaluation using combined scores of staining intensity and percentage of positive cells provides a standardized approach. In colon adenocarcinoma research, staining intensity (0-3) multiplied by positive cell percentage score (0-4) with scores >3 classified as positive has been effective .

• Digital image analysis: Software-based quantification of immunohistochemically stained slides can provide more objective and reproducible measurements of GIPC2 expression compared to manual scoring.

How does GIPC2 interact with signaling pathways in different cancer types?

GIPC2 demonstrates complex interactions with multiple signaling pathways in a cancer type-specific manner. In prostate cancer, GIPC2 interacts with Fzd7 to promote cancer metastasis through Wnt signaling pathways . Ingenuity Pathway Analysis (IPA) network and upstream analysis demonstrated that many GIPC2-associated effectors are involved in WNT-related pathways . In colon adenocarcinoma, Gene Set Enrichment Analysis (GSEA) revealed that GIPC2 expression is enriched in 'cell cycle checkpoints', 'DNA replication', and 'mitosis-associated signaling pathways' . These diverse pathway interactions highlight the context-dependent nature of GIPC2 function, requiring careful experimental design when using antibodies to study these mechanisms.

What is the relationship between GIPC2 expression and immune cell infiltration in cancer microenvironments?

The relationship between GIPC2 expression and immune cell infiltration varies by cancer type. In colon adenocarcinoma, high GIPC2 expression is positively associated with increased levels of infiltrating immune cells . This relationship can be studied using antibody-based methods combined with computational approaches. The CIBERSORT algorithm can estimate the presence of different immune cell populations, and these predictions can be validated using multiplexed immunohistochemistry with GIPC2 antibodies alongside immune cell markers. Additionally, the expression of immune checkpoint-associated genes (CD274, CTLA4, HAVCR2, LAG3, PDCD1, PDCD1LG2, TIGIT, and SIGLEC15) is significantly higher in groups with low GIPC2 expression in COAD , suggesting a complex relationship between GIPC2 expression and immune regulation.

How can researchers effectively combine GIPC2 antibody-based detection with transcriptomic analysis?

An integrated approach combining antibody-based detection with transcriptomic analysis provides comprehensive insights into GIPC2 function. Researchers should:

• Validate transcript-level findings at the protein level by correlating RNA-seq or microarray data with GIPC2 antibody-based detection in the same samples.

• Use cell lines with GIPC2 overexpression or knockdown to identify genes co-regulated with GIPC2, then validate these relationships in clinical samples using multiplexed immunohistochemistry.

• Employ tools like GEPIA2 to estimate the top genes co-expressed with GIPC2, followed by enrichment analysis using the clusterProfiler package for GO biological process and KEGG pathway analysis .

• Validate key relationships using Spearman rank correlation tests between GIPC2 and top co-expressed genes , then confirm these correlations at the protein level using antibody-based methods.

How can GIPC2 antibody-based assays be developed for potential diagnostic applications?

Development of GIPC2 antibody-based diagnostic assays requires careful consideration of cancer type-specific expression patterns. For prostate cancer, where GIPC2 expression is increased in metastatic disease (mean relative expression 280.06 ± 240.1 in metastatic vs. 41.36 ± 16.81 in primary tumors) , assays could focus on detecting elevated GIPC2 as a potential metastasis biomarker. For colon adenocarcinoma, where GIPC2 is downregulated compared to normal tissue but high expression correlates with better prognosis , diagnostic approaches would differ. Development steps include: (1) Antibody validation across tissue types; (2) Establishment of expression thresholds through ROC curve analysis; (3) Correlation with clinical outcomes in prospective studies; (4) Integration with existing biomarker panels; (5) Standardization of detection protocols across laboratories. Such assays could potentially help stratify patients for treatment decisions or monitoring purposes.

What experimental approaches can be used to study GIPC2's role in cancer metastasis?

Multiple experimental approaches can elucidate GIPC2's role in cancer metastasis:

• In vitro invasion and migration assays: Transwell-based invasion and migration assays with GIPC2 overexpression or knockdown can quantitatively evaluate cellular invasion, as demonstrated in RWPE-1 cells where GIPC2 overexpression increased invasion .

• Microfluidic assays: These can evaluate multiple aspects of metastasis including adhesion, invasion, and migration, with cell quantification across multiple imaging layers .

• In vivo metastasis models: Cardiac injection of cancer cells with altered GIPC2 expression into male athymic nude mice followed by bioluminescence monitoring and ex vivo imaging provides comprehensive metastasis assessment .

• Mechanistic studies: Combining the above approaches with pathway inhibitors or co-immunoprecipitation using GIPC2 antibodies can identify key interaction partners, such as the demonstrated interaction between GIPC2 and Fzd7 in prostate cancer .

• Clinical correlation: Validating experimental findings through GIPC2 antibody staining in patient samples with known metastatic status establishes clinical relevance of laboratory findings .