GIPC2 Antibody

What is GIPC2 and what are its key cellular functions?

GIPC2 is a member of the GIPC family of adaptor proteins containing a central PDZ domain. It functions as a scaffold protein that interacts with various binding partners to regulate multiple biological processes. Research demonstrates that GIPC2 participates in:

• Cell signaling pathway regulation

• Transmembrane protein transport

• Cell movement and endocytosis

• Cell cycle checkpoints via p27 regulation through NONO interaction

• DNA replication and mitosis-associated signaling pathways

GIPC2 serves an important role in the development of digestive tract tumors and appears to function as a tumor suppressor in several cancer types, including colon adenocarcinoma (COAD) .

What is the molecular weight of GIPC2 protein when detected by Western blot?

GIPC2 protein has a calculated molecular weight of 34 kDa based on its 315 amino acid sequence. When detected by Western blot, researchers should expect to observe a band at approximately 34 kDa . This consistent molecular weight has been observed across multiple antibody sources and experimental conditions. When planning Western blot experiments, it's advisable to include positive control samples such as HEK-293 cells, MKN-45 cells, T-47D cells, or mouse colon tissue, which have been validated to show detectable GIPC2 expression .

Which tissues normally express high levels of GIPC2?

GIPC2 shows tissue-specific expression patterns that are important for experimental design and control selection:

When designing experiments, normal colon tissue samples represent ideal positive controls for GIPC2 expression studies, while colorectal cancer tissues can serve as comparative samples with typically lower expression levels .

How should GIPC2 antibodies be validated for immunohistochemistry applications?

Thorough validation of GIPC2 antibodies for immunohistochemistry involves multiple steps:

• Positive control selection: Use normal adult kidney tissue and normal colon tissue, which show reliable GIPC2 expression .

• Negative controls: Include PBS instead of primary antibody as a procedural negative control to assess non-specific binding.

• Antibody dilution optimization: While specific IHC dilutions vary by manufacturer, most protocols recommend initial titration experiments testing a range of dilutions (e.g., 1:100 to 1:500) .

• Cross-validation methodology: Compare IHC results with Western blot and qRT-PCR data from the same samples to confirm expression patterns across techniques.

• Scoring system development: Based on published research, establish a consistent scoring system. For example, in COAD studies, researchers classified samples as GIPC2-positive or GIPC2-negative based on established criteria .

• Documentation of staining patterns: GIPC2 staining should be evaluated for subcellular localization, as some studies suggest nuclear localization predominates in certain contexts .

Implementing this comprehensive validation approach ensures reliable and reproducible GIPC2 detection in tissue samples.

What expression differences are observed between normal and cancerous tissues?

Research demonstrates consistent GIPC2 expression differences between normal and cancerous tissues:

• Colorectal cancer: GIPC2 expression is significantly downregulated in colon adenocarcinoma compared to normal colon tissues (p<0.05) .

• Quantitative differences: IHC analysis revealed that the positive rate of GIPC2 in normal intestinal mucosa (81.82%, 18/22) was significantly higher than in COAD samples (13.64%, 3/22, χ²=20.497, P<0.001) .

• Cell line evidence: GIPC2 expression is lower in colorectal cancer cell lines (Lovo, RKO, DLD-1, HCT116) compared to normal colon epithelial cells (HcoEpiC) .

• Pheochromocytoma: GIPC2 exhibits reduced expression and functions as a putative tumor suppressor gene .

• Prostate cancer: Interestingly, GIPC2 shows a different pattern, with increased expression in metastatic prostate tumors compared to localized tumors or normal prostate cells .

These tissue-specific differences highlight the importance of cancer-specific analysis rather than generalizing findings across cancer types.

How can researchers address contradictory findings regarding GIPC2 expression across cancer types?

Resolving contradictory findings regarding GIPC2 expression requires a systematic approach:

• Conduct tissue-specific analyses: Given that GIPC2 shows opposite expression patterns in colorectal cancer (downregulated) versus metastatic prostate cancer (upregulated) , researchers should avoid generalizing across cancer types.

• Stratify by stage and grade: Expression patterns may vary by cancer progression stage. For instance, in prostate cancer, GIPC2 expression differs between localized and metastatic disease .

• Employ multi-omics integration:

  • Analyze promoter methylation (shown to influence GIPC2 expression)

  • Compare mRNA and protein expression levels

  • Assess copy number variations

  • Examine pathway activation differences

• Functional validation: Perform knockdown and overexpression studies in multiple cell lines from each cancer type to verify functional effects, as demonstrated in prostate cancer and pheochromocytoma studies .

• Pathway context analysis: Different pathway involvement may explain contradictory roles. For example, GIPC2 activates the WNT-β-catenin pathway in prostate cancer but may act through different mechanisms in colorectal cancer.

This comprehensive approach can help reconcile apparently contradictory findings and develop a more nuanced understanding of GIPC2's context-dependent roles.

What signaling pathways does GIPC2 participate in based on current evidence?

GIPC2 participates in several key signaling pathways with cancer relevance:

• Cell Cycle Regulation: GIPC2 regulates cell cycle gene p27 through interaction with NONO, a nucleoprotein involved in cell cycle regulation . Gene Set Enrichment Analysis shows enrichment in "cell cycle checkpoints" pathways .

• DNA Replication and Mitosis: GIPC2 expression is associated with DNA replication and mitosis-associated pathways .

• Intestinal Function Pathways: Co-expression analysis revealed enrichment in pathways related to:

  • Intestinal absorption

  • Regulation of microvillus organization

  • Microvillus organization

• Epithelial Cell Functions: GO analysis shows significant enrichment in:

  • Epithelial cell signaling

  • Tight junction

  • Brush border

  • Apical junction complex

• WNT-β-catenin Pathway: In prostate cancer, GIPC2 activates the WNT-β-catenin pathway. GIPC2 overexpression leads to β-catenin accumulation, which can be blocked by DKK1 (a WNT signaling inhibitor) .

• PI3K/AKT Pathway: GIPC2 has been implicated in regulating the PI3K/AKT pathway, particularly in the context of acute myeloid leukemia .

This diverse pathway involvement may explain GIPC2's complex roles across different cancer types.

How can GIPC2 antibodies be used to study immune cell infiltration in tumors?

GIPC2 antibodies can provide valuable insights into tumor immune microenvironment:

• Expression correlation with immune cell populations: High GIPC2 expression is associated with increased numbers of:

  • Plasma B cells (P=0.018)

  • Resting CD4+ memory T cells (P=0.015)

  • Activated CD4+ memory T cells (P=0.023)

  • Activated myeloid dendritic cells (P=0.005)

  • Activated mast cells (P=0.023)

  And decreased numbers of:

  • Regulatory T cells (P=0.021)

  • M0 macrophages (P=0.038)

  • Neutrophils (P=0.029)

• Multiplex immunostaining methodology: Combine GIPC2 antibodies with immune cell markers in multiplex immunofluorescence to visualize spatial relationships between GIPC2-expressing cells and immune infiltrates.

• Immune checkpoint relationship investigation: GIPC2 expression levels correlate with immune checkpoint gene expression (HAVCR2, LAG3, PDCD1, SIGLEC15), which were significantly higher in low GIPC2 expression groups .

• Experimental design approach:

  • Stratify samples by GIPC2 expression (high vs. low)

  • Quantify immune cell populations using algorithms like CIBERSORT

  • Analyze correlations between GIPC2 expression and immune profiles

  • Validate using immunohistochemistry with appropriate controls

This integrated approach can reveal mechanisms by which GIPC2 influences the tumor immune microenvironment, potentially informing immunotherapeutic strategies.

What are the challenges and solutions in detecting low levels of GIPC2 in tissue samples?

Detecting low GIPC2 levels, particularly in cancer tissues, presents several challenges with corresponding solutions:

For extreme cases of low expression, consider using cell models treated with demethylating agents like DAC (as demonstrated in prostate cancer cell lines), which can restore GIPC2 expression by decreasing methylation levels .

What co-expressed genes and interacting proteins should be considered in GIPC2 research?

Research has identified several key GIPC2-associated genes and proteins:

• Top co-expressed genes in colorectal cancer:

  • EPCAM

  • LRRC8D

  • EPB41L4B

  • ACSL5

  • CDS1

  These genes show significant positive correlation with GIPC2 expression and many are involved in maintaining normal intestinal mucosal function and cancer resistance .

• Verified interacting proteins:

  • NONO: A nucleoprotein with a known role in cell cycle regulation; GIPC2 regulates cell cycle gene p27 through NONO

  • Fzd7: GIPC2 interacts with Fzd7 in prostate cancer to promote metastasis

• Pathway-related proteins:

  • β-catenin: GIPC2 overexpression leads to β-catenin accumulation in prostate cancer cells

  • GSK-3β: GIPC2 affects GSK-3β phosphorylation status

  • Immune checkpoint proteins: HAVCR2, LAG3, PDCD1, and SIGLEC15 show expression correlation with GIPC2

When designing co-IP or other interaction studies, these proteins represent high-priority candidates for investigation. Mass spectrometry approaches, as used in the identification of the GIPC2-NONO interaction , can further expand our understanding of the GIPC2 interactome.

What is the prognostic significance of GIPC2 expression in cancer?

GIPC2 expression shows significant prognostic value in several cancer types:

These findings collectively indicate that GIPC2 expression could serve as a valuable prognostic biomarker, particularly in colorectal cancer, where high expression correlates with improved patient outcomes.

How can dual staining with GIPC2 and other markers be optimized for studying pathway interactions?

Optimizing dual staining with GIPC2 and other markers requires careful methodological considerations:

• Marker selection based on pathway evidence:

  • Cell cycle markers: p27, cyclins, CDKs (based on GIPC2's regulation of p27)

  • WNT-β-catenin pathway: β-catenin, GSK-3β (based on GIPC2's activation of this pathway)

  • Immune checkpoint markers: HAVCR2, LAG3, PDCD1, SIGLEC15 (shown to correlate with GIPC2 expression)

  • Co-expressed genes: EPCAM, LRRC8D, EPB41L4B, ACSL5, CDS1

• Technical optimization protocol:

  • Use primary antibodies from different host species (rabbit anti-GIPC2 paired with mouse antibodies against other markers)

  • For immunofluorescence, select fluorophores with minimal spectral overlap

  • For brightfield IHC, use contrasting chromogens (e.g., DAB and Fast Red)

  • Include single-stained controls alongside dual staining

• Advanced visualization techniques:

  • Employ confocal microscopy for detailed co-localization analysis

  • Use digital image analysis software to quantify co-expression patterns

  • Apply spatial statistics to analyze proximity relationships between markers

• Validation approach:

  • Complement imaging with biochemical interaction studies (co-IP)

  • Use GIPC2 knockdown or overexpression models to confirm functional relevance

  • Include appropriate tissue controls with known expression patterns

This systematic approach enables robust investigation of GIPC2's interactions with various pathways and proteins, potentially revealing new therapeutic targets or biomarkers.

What are the appropriate experimental controls when studying GIPC2 in relation to immune checkpoint inhibitors?

When investigating GIPC2 in relation to immune checkpoint inhibitors, comprehensive controls are essential:

• Expression level controls:

  • Positive control tissues: Normal colon tissue (high GIPC2 expression)

  • Negative control tissues: Colon adenocarcinoma with verified low GIPC2 expression

  • Cell line panel controls: Normal colon epithelial cells (HcoEpiC) versus CRC cell lines (Lovo, RKO, DLD-1, HCT116)

• Experimental manipulation controls:

  • GIPC2 modulation models: Include both knockdown and overexpression systems

  • Vector controls: Empty vector controls for overexpression studies

  • siRNA controls: Non-targeting siRNA sequences for knockdown experiments

• Immune checkpoint-specific controls:

  • Treatment conditions: Include samples with and without immune checkpoint inhibitors

  • Antibody controls: Use isotype-matched control antibodies

  • Response model controls: Include known responder and non-responder models

• Validation methodology:

  • Multi-technique verification: Combine protein detection (IHC, WB) with mRNA analysis

  • Cellular localization assessment: Verify whether GIPC2 expression changes affect cellular distribution of immune checkpoint proteins

  • Functional assays: Include T-cell activation assays to measure functional consequences of GIPC2 modulation

Given the established relationship between GIPC2 expression and immune checkpoint genes , these controls will help elucidate mechanisms by which GIPC2 might influence immunotherapy response in cancer.

How does GIPC2 expression influence epithelial-mesenchymal transition in cancer?

Emerging research indicates GIPC2 may regulate epithelial-mesenchymal transition (EMT):

Recent studies have identified GIPC2 as part of a prognostic signature correlated with epithelial-mesenchymal transition in colorectal cancer. Experimental evidence demonstrates that GIPC2 overexpression inhibits the malignant characteristics of CRC cells through upregulating E-cadherin while affecting Vimentin and Snail expression, key markers in the EMT process .

Conversely, GIPC2 knockdown promotes the opposite effect in CRC cells, suggesting GIPC2 functions as an EMT regulator. This mechanism may partially explain how GIPC2 influences cancer progression and metastasis, making it a promising clinical biomarker or therapeutic target for CRC .

Future studies should explore the specific molecular mechanisms by which GIPC2 regulates EMT-related proteins and whether this function is consistent across different cancer types or context-dependent.

What is the relationship between GIPC2 promoter methylation and expression in cancer?

The relationship between GIPC2 promoter methylation and expression represents an important epigenetic regulatory mechanism:

In prostate cancer studies, researchers have identified significant negative correlation (p<0.01) between GIPC2 promoter methylation and expression levels . Treatment of various cell lines (including RWPE-1, C4-2, and Du145) with the DNMT1 inhibitor DAC decreased methylation levels and significantly increased GIPC2 mRNA and protein expression .

DNA sequencing confirmed CpG-island methylation at the GIPC2 promoter, and time-course experiments demonstrated gradual increases in GIPC2 protein expression following demethylation treatment .

This epigenetic regulation may explain tissue-specific and cancer-specific expression patterns of GIPC2, suggesting that demethylating agents might restore GIPC2 expression in cancers where it functions as a tumor suppressor. Future research should investigate whether similar methylation patterns regulate GIPC2 in colorectal and other cancers, potentially opening avenues for epigenetic therapies.