ARHGAP4 Antibody

What is ARHGAP4 and why is it important in cellular research?

ARHGAP4, also known as Rho GTPase-activating protein 4 or Rho-GAP hematopoietic protein C1, functions as a regulator of Rho family GTPases. It has an inhibitory effect on stress fiber organization and may down-regulate Rho-like GTPase in hematopoietic cells . Its importance in research stems from its involvement in cellular signaling pathways that regulate cell migration, invasion, and other cancer-related processes. Recent studies have demonstrated its roles in colorectal cancer, kidney renal cancer, and pancreatic cancer, making it a potential biomarker and therapeutic target .

What are the validated applications for ARHGAP4 antibodies?

ARHGAP4 antibodies have been validated for several research applications, including:

• Western blotting (WB) for protein expression analysis

• Immunoprecipitation (IP) for protein complex studies

• Immunohistochemistry (IHC) for tissue localization studies

The antibodies have been particularly validated for detecting human ARHGAP4, though cross-reactivity with other species may be possible based on sequence homology .

What is the optimal protocol for immunohistochemical detection of ARHGAP4 in tissue samples?

For optimal immunohistochemical detection of ARHGAP4 in tissue samples, researchers should follow this methodological approach:

• Tissue fixation in 4% paraformaldehyde followed by paraffin embedding

• Section tissues into approximately 5-μm-thick slices

• Perform antigen retrieval using solution low pH (such as Dako K8005) at 90°C for 20 minutes

• Wash three times in phosphate-buffered saline (PBS)

• Block endogenous peroxidase activity with 0.3% hydrogen peroxide and 1% methanol in PBS for 10 minutes

• Block with 5% normal goat serum for 1 hour

• Incubate with anti-ARHGAP4 antibody (1:200 dilution) overnight at 4°C

• Incubate with biotinylated secondary antibody (such as goat anti-rabbit, 1:200 dilution) for 1 hour

• Apply Avidin/biotin complex for 30 minutes

• Visualize using chromogen Diaminobenzidine (DAB)

• Counterstain nuclei with hematoxylin

This protocol has been successfully used in studies examining ARHGAP4 expression in cancer tissues.

How should ARHGAP4 antibody specificity be validated in experimental studies?

Validating ARHGAP4 antibody specificity is critical for research reliability. A comprehensive validation approach should include:

• Positive and negative controls: Use tissues or cell lines known to express high levels of ARHGAP4 (such as Bxpc3 cells) as positive controls and compare with those with minimal expression

• Knockout validation: Compare staining between wild-type and ARHGAP4 knockout or knockdown samples to confirm specificity

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples; this should abolish specific staining

• Molecular weight verification: Confirm that the detected band in Western blot corresponds to the expected molecular weight of ARHGAP4

• Multiple antibody comparison: When possible, use different antibodies targeting distinct epitopes of ARHGAP4 to verify consistent results

What are the optimal storage conditions for maintaining ARHGAP4 antibody functionality?

To maintain ARHGAP4 antibody functionality over time, researchers should:

• Store stock solutions at -20°C in small aliquots to minimize freeze-thaw cycles

• For short-term storage (1-2 weeks), keep working dilutions at 4°C with preservatives like sodium azide (0.02%)

• Avoid repeated freeze-thaw cycles as they can lead to antibody denaturation and loss of binding capacity

• Prior to use, centrifuge the antibody solution briefly to bring down any precipitates

• Follow manufacturer-specific guidelines, as different formulations may have unique storage requirements

How is ARHGAP4 expression associated with colorectal cancer prognosis?

Interestingly, while ARHGAP4 is highly expressed in READ (rectal adenocarcinoma) compared to normal tissues, the difference in OS and DFS between high and low expression groups was not statistically significant for READ . These findings suggest tissue-specific prognostic value of ARHGAP4 expression within different subtypes of colorectal cancer.

What is the relationship between ARHGAP4 expression and immune cell infiltration in cancer?

ARHGAP4 expression shows significant correlations with immune cell infiltration in cancer, particularly in colorectal cancer. Research has demonstrated that:

• ARHGAP4 expression is highly correlated with CD4+ T-cell infiltration in colorectal cancer (CRC) and with dendritic cell infiltration specifically in rectal adenocarcinoma (READ)

• In colon cancer, ARHGAP4 gene knockout leads to downregulation of B cells, macrophages, neutrophils, and dendritic cells

• Conversely, high amplification of the ARHGAP4 gene is associated with upregulation of CD8+ and CD4+ T cells, neutrophils, and dendritic cells in colon adenocarcinoma (COAD)

• In kidney renal cell carcinoma (KIRC), ARHGAP4 expression is positively correlated with cytotoxic cells

These findings suggest ARHGAP4 may play important roles in modulating the tumor immune microenvironment, with potential implications for immunotherapy approaches.

How does ARHGAP4 interact with the SEPT2-SEPT9 complex and what are the functional implications?

ARHGAP4 forms a complex with SEPT2 and SEPT9, which has significant functional implications. Research has confirmed through co-immunoprecipitation that GFP-ARHGAP4-associated complex contains both SEPT2 and SEPT9 . This interaction has been verified by pulling down with SEPT2 and SEPT9 antibodies and confirming association with ARHGAP4 in both cases .

The functional implications of this interaction include:

• Regulation of focal adhesions (FAs): The ARHGAP4-SEPT2-SEPT9 complex influences FA dynamics, with ARHGAP4 knockdown leading to an increase in the total number of FAs, particularly small patches less than 1.5 μm² in area

• Cell morphology regulation: Silencing of ARHGAP4 causes morphological changes in cells, giving them a spread morphology compared to control cells

• Protein expression modulation: Overexpression of ARHGAP4 reduces paxillin protein expression, while deletion mutants of specific ARHGAP4 domains (particularly Rho-GAP and SH3 domains) reverse this effect

• Bidirectional regulation: The complex enables both up- and down-regulation of focal adhesions, suggesting a sophisticated regulatory mechanism

What are the critical controls needed when assessing ARHGAP4 protein levels in cancer tissues?

When assessing ARHGAP4 protein levels in cancer tissues, the following critical controls should be implemented:

• Adjacent normal tissue controls: Always include matched adjacent normal tissue samples from the same patients to establish baseline expression levels and account for individual variation

• Positive tissue controls: Include samples known to express high levels of ARHGAP4 (such as rectal adenocarcinoma tissues or Bxpc3 cell line derived tumors) as positive controls

• Negative controls: Generate by omitting the primary antibody while maintaining all other steps in the protocol to assess non-specific binding of secondary antibodies

• Isotype controls: Include an irrelevant antibody of the same isotype to evaluate non-specific binding

• Expression cut-off validation: Establish and validate cut-off points for high versus low expression (e.g., a minimum of 10% of tumor epithelial cells showing positive staining)

• Blind assessment: Have samples assessed by experienced pathologists who are blinded from outcome data to prevent bias

• Secondary validation method: Confirm immunohistochemistry findings with a secondary method such as Western blotting or RT-PCR when possible

How should researchers approach conflicting data regarding ARHGAP4's role in different cancer types?

When confronting conflicting data regarding ARHGAP4's role across different cancer types, researchers should:

• Acknowledge tissue-specific effects: ARHGAP4 shows different prognostic significance in COAD versus READ, suggesting cancer subtype-specific roles . Similarly, its immune infiltration correlations vary between cancer types

• Consider molecular context: Evaluate the expression and activation status of ARHGAP4's interaction partners (like SEPT2 and SEPT9) in each cancer type, as these may modulate its function

• Assess methodological differences: Compare antibody clones, detection methods, scoring systems, and cut-off thresholds used across studies

• Integrate multi-omics data: Combine protein expression data with genomic alterations, transcriptomics, and epigenetic information to build a more comprehensive understanding

• Conduct pathway analysis: Determine if ARHGAP4 is functioning through different signaling pathways in different cancers (e.g., HDAC2/β-Catenin in pancreatic cancer versus other pathways in colorectal cancer)

• Design comparative studies: Directly compare ARHGAP4 function across multiple cancer cell lines under identical experimental conditions

• Consider clinical heterogeneity: Account for differences in patient cohorts, treatment histories, and cancer stages when comparing results across studies

What experimental approaches can determine the causal relationship between ARHGAP4 expression and immune cell infiltration?

To establish a causal relationship between ARHGAP4 expression and immune cell infiltration, researchers should consider these experimental approaches:

• In vivo ARHGAP4 manipulation models:

  • Generate conditional knockout or overexpression mouse models of ARHGAP4 in specific cancer types

  • Use inducible systems to modulate ARHGAP4 expression at different stages of tumor development

  • Analyze immune cell infiltration through flow cytometry, immunohistochemistry, and single-cell RNA sequencing

• Co-culture systems:

  • Establish co-culture systems between ARHGAP4-manipulated cancer cells and immune cells

  • Use transwell migration assays to assess if ARHGAP4 expression affects immune cell chemotaxis

  • Analyze changes in cytokine/chemokine production profiles

• Mechanistic investigations:

  • Identify ARHGAP4-dependent secretory factors using proteomics

  • Perform chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors regulated by ARHGAP4 that control immune-related genes

  • Investigate if ARHGAP4's GAP activity toward specific Rho GTPases mediates immune cell recruitment

• Clinical validation:

  • Correlate ARHGAP4 genetic alterations with immune signature scores in large patient cohorts

  • Analyze if ARHGAP4 expression predicts response to immunotherapy

  • Perform spatial transcriptomics to map the relationship between ARHGAP4-expressing cells and immune niches within tumors

What are common challenges in detecting ARHGAP4 in clinical samples and how can they be addressed?

Common challenges in detecting ARHGAP4 in clinical samples include:

• Tissue fixation variability: Prolonged formalin fixation can mask epitopes

  • Solution: Optimize antigen retrieval methods (try both heat-induced epitope retrieval at pH 6.0 and pH 9.0)

  • Consider testing enzyme-based retrieval for challenging samples

• Low signal intensity: ARHGAP4 may be expressed at low levels in some tissues

  • Solution: Use signal amplification systems like tyramide signal amplification

  • Increase primary antibody concentration or incubation time (overnight at 4°C)

  • Try higher sensitivity detection systems such as polymer-HRP systems

• Background staining: Non-specific binding can obscure specific signals

  • Solution: Increase blocking time or concentration (try 5-10% normal serum)

  • Use additional blocking agents such as bovine serum albumin

  • Include avidin/biotin blocking steps if using biotin-based detection systems

• Inconsistent results between sample types:

  • Solution: Standardize pre-analytical variables including time to fixation

  • Develop tissue-specific protocols for different cancer types

  • Use positive control tissues with each run to ensure consistency

How should researchers optimize Western blot protocols for detecting ARHGAP4?

For optimal Western blot detection of ARHGAP4, researchers should:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors for cell/tissue lysis

  • Include phosphatase inhibitors to preserve phosphorylation states

  • Sonicate samples briefly to shear DNA and reduce viscosity

• Protein separation:

  • Use 8-10% SDS-PAGE gels as ARHGAP4 has a molecular weight of approximately 115 kDa

  • Run gels at lower voltage (80-100V) for better resolution

• Transfer optimization:

  • Use wet transfer at 30V overnight at 4°C for large proteins like ARHGAP4

  • Consider adding SDS (0.1%) to transfer buffer to improve transfer efficiency

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat dry milk in TBST for 1-2 hours

  • Incubate with anti-ARHGAP4 antibody (1:500-1:1000 dilution) overnight at 4°C

  • Perform extensive washing steps (at least 3 times for 10 minutes each)

  • Use HRP-conjugated secondary antibodies at 1:5000-1:10000 dilution

• Detection considerations:

  • Use enhanced chemiluminescence (ECL) detection for standard applications

  • Consider more sensitive detection methods such as ECL Plus for low expression samples

  • Include positive control lysates from cells known to express ARHGAP4 (like HEK293 or Bxpc3 cells)

What strategies can enhance the reproducibility of ARHGAP4 immunoprecipitation experiments?

To enhance reproducibility in ARHGAP4 immunoprecipitation experiments, researchers should implement these strategies:

• Lysis optimization:

  • Use gentle lysis buffers that preserve protein complexes (e.g., 1% NP-40 or 0.5% Triton X-100)

  • Include protease and phosphatase inhibitors freshly before use

  • Maintain samples at 4°C throughout processing

• Antibody selection and validation:

  • Test multiple antibodies targeting different epitopes of ARHGAP4

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Include isotype control antibodies as negative controls

• Binding and washing conditions:

  • Optimize antibody-to-lysate ratio (typically 2-5 μg antibody per 500 μg protein)

  • Extend binding time (overnight at 4°C) to maximize interaction

  • Use a series of increasingly stringent washes to remove non-specific binders

• Complex preservation:

  • Consider crosslinking reagents for transient interactions

  • Use staged elution methods to distinguish strong versus weak interactors

  • When studying ARHGAP4-SEPT2-SEPT9 complexes, pull down with antibodies against each component in parallel experiments for validation

• Detection optimization:

  • Use gradient gels for better separation of complex components

  • Consider silver staining for detecting low-abundance interacting partners

  • Follow with mass spectrometry analysis to identify novel interaction partners