rhpn2 Antibody

What is RHPN2 and why is it important in cancer research?

RHPN2 (Rhophilin Rho GTPase-binding protein 2) is a 77 kDa protein that binds specifically to GTP-Rho. It plays crucial roles in cellular processes involving cytoskeletal dynamics and has been identified as a significant factor in cancer progression. RHPN2 functions in Rho pathways to regulate stress fiber formation and F-actin structure turnover .

Its importance in cancer research stems from findings that RHPN2 amplification/overexpression correlates with decreased survival in various cancer types. For example, in glioblastoma, RHPN2 amplification has been identified as a central genetic determinant of the mesenchymal phenotype that correlates with dramatically decreased patient survival . Similarly, in lung adenocarcinoma, high RHPN2 expression is associated with poor prognosis and promotes tumorigenesis through various mechanisms .

What applications are RHPN2 antibodies most commonly used for?

RHPN2 antibodies are validated for multiple applications in molecular and cellular biology research:

When selecting an antibody for your research, it's important to choose one validated for your specific application and species of interest. Many commercial antibodies show reactivity with human, mouse, and rat samples .

How should RHPN2 antibodies be stored and handled to maintain optimal activity?

For optimal antibody performance, proper storage and handling are essential:

• Store RHPN2 antibodies at -20°C in their recommended buffer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Most antibodies remain stable for one year after shipment when properly stored

• Aliquoting is generally unnecessary for -20°C storage of small volumes

• Some formulations contain 0.1% BSA as a stabilizer, particularly in smaller volumes (20μl)

• Avoid repeated freeze-thaw cycles

• When diluting, use fresh, cold buffer and maintain sterile conditions

• Check the antibody datasheet for specific recommendations, as formulations may vary between manufacturers

What are the optimal conditions for detecting RHPN2 in tissue samples by immunohistochemistry?

Based on validated protocols, the following methodology is recommended for RHPN2 detection in tissue samples:

• Fixation: Formalin-fixed, paraffin-embedded tissue sections are commonly used

• Antigen retrieval: Use TE buffer pH 9.0 as the primary method; alternatively, citrate buffer pH 6.0 may be used

• Blocking: Block with appropriate serum (typically 5-10% normal serum from the same species as the secondary antibody)

• Primary antibody: Incubate with anti-RHPN2 antibody at dilutions of 1:50-1:500 (optimize for each tissue type)

• Detection: Use appropriate detection system (HRP/DAB or fluorescent-based)

• Controls: Always include positive controls (colon or stomach tissue show reliable RHPN2 expression) and negative controls

For fluorescent detection, the signal can be visualized using appropriate secondary antibodies such as CoraLite® Plus 488-Goat Anti-Rabbit secondary antibodies .

What experimental controls should be implemented when studying RHPN2 expression in cancer tissues?

Robust experimental design requires multiple controls:

• Tissue controls:

  • Positive tissue controls: Use tissues known to express RHPN2 (colon, stomach)

  • Negative tissue controls: Use tissues with minimal RHPN2 expression

  • Matched normal adjacent tissue from the same patient

• Antibody controls:

  • Primary antibody omission control

  • Isotype control antibody

  • Blocking peptide competition assay to confirm specificity

• Expression validation controls:

  • Correlate protein expression with mRNA expression (RT-qPCR)

  • Use multiple antibodies targeting different epitopes of RHPN2

  • Include RHPN2 knockdown/knockout samples if available

• Cancer-specific considerations:

  • Include samples from different tumor grades/stages

  • Compare expression patterns across tumor subtypes

  • Use cell lines with known RHPN2 expression levels (HepG2, COLO 320) as reference points

How can researchers effectively validate RHPN2 antibody specificity?

Antibody validation is critical for reliable research results. A comprehensive validation approach should include:

• Western blot validation:

  • Confirm single band at expected molecular weight (77 kDa for RHPN2)

  • Test in multiple cell lines with varying expression levels

  • Include positive controls (COLO 320, HepG2 cells)

• Genetic approaches:

  • Test antibody in RHPN2 knockdown (siRNA/shRNA) or knockout (CRISPR) samples

  • Perform rescue experiments with ectopic RHPN2 expression

• Epitope mapping:

  • Use antibodies targeting different regions of RHPN2 (N-terminal, central, C-terminal)

  • Compare detection patterns across techniques

• Orthogonal validation:

  • Correlate protein detection with mRNA levels using RT-qPCR

  • Consider mass spectrometry-based validation for absolute confirmation

• Cross-reactivity assessment:

  • Test in samples containing related proteins (RHPN1, other Rho-binding proteins)

  • Evaluate species cross-reactivity if working with non-human models

How does RHPN2 contribute to the mesenchymal transformation in glioblastoma?

RHPN2 has been identified as a central genetic determinant of the mesenchymal (MES) phenotype in glioblastoma (GBM), which is associated with increased aggressiveness and poor patient outcomes. The mechanistic pathway involves:

• RHPN2 amplification/overexpression:

  • Genomic amplification of RHPN2 on chromosome 19 correlates with decreased survival in glioma patients

  • Amplification is detected predominantly in the MES subtype of GBM

• Cellular effects:

  • Ectopic expression of RHPN2 in neural stem cells and astrocytes triggers expression of MES-specific genes

  • RHPN2 expression promotes invasive phenotypes without affecting cell proliferation

  • Cells acquire elongated, spindle-shaped morphology characteristic of mesenchymal transformation

• Molecular mechanism:

  • RHPN2 activates RhoA, a master regulator of cell migration and invasion

  • This activation increases RhoA-GTP levels by more than 4-fold in primary human astrocytes

  • The RHPN2-RhoA pathway modulates cytoskeletal dynamics, promoting migration and invasion

• Functional consequences:

  • Enhanced invasion through extracellular matrix (demonstrated in Matrigel invasion assays)

  • Increased cell migration (shown in wound healing assays)

  • No significant effect on cell proliferation rates

These findings indicate that RHPN2 amplification specifically drives the mesenchymal transformation that contributes to the worst clinical outcomes in GBM patients .

What is the relationship between RHPN2 expression and glutamine metabolism in lung cancer?

Recent research has uncovered a novel connection between RHPN2 and glutamine metabolism in lung adenocarcinoma (LUAD):

This metabolic reprogramming represents a significant mechanism by which RHPN2 contributes to lung cancer progression and provides potential targets for therapeutic intervention .

How do the roles of RHPN2 differ between hepatocellular carcinoma and glioblastoma?

RHPN2 exhibits both common and distinct roles across different cancer types:

These differences highlight the context-dependent functions of RHPN2 in cancer biology and suggest that therapeutic strategies targeting RHPN2 may need to be tailored to specific cancer types .

How can researchers address the apparent contradictions in RHPN2's effect on RhoA activity across different studies?

The literature shows conflicting results regarding RHPN2's effect on RhoA activity, which requires careful consideration:

• Documented contradictions:

  • In glioblastoma studies, RHPN2 was reported to activate RhoA, increasing RhoA-GTP levels

  • In lung cancer research, RHPN2 was found to attenuate RhoA activity and block serum-induced RhoA activation

• Possible explanations:

  • Cell type-specific effects: Different cell types (neural cells vs. lung epithelial cells) may have distinct RhoA regulatory networks

  • Context-dependent function: RHPN2 may have opposing effects depending on cellular context, activation state, or microenvironment

  • Protein isoforms or mutations: Different RHPN2 variants or mutations might differentially affect RhoA activity

  • Experimental conditions: Variations in experimental conditions (serum levels, growth factors) may influence outcomes

• Methodological approach to resolve contradictions:

  • Compare RhoA activity using the same assay system across cell types

  • Include appropriate positive and negative controls

  • Perform domain mapping to identify regions of RHPN2 responsible for differential effects

  • Use point mutants (e.g., V73M within the Rho-binding domain) to dissect specific interactions

  • Examine downstream effectors of RhoA to confirm functional outcomes

  • Consider temporal dynamics of RHPN2-RhoA interactions

What are the key considerations when interpreting RHPN2 immunohistochemistry results in clinical samples?

Accurate interpretation of RHPN2 immunohistochemistry requires attention to several factors:

• Staining pattern evaluation:

  • RHPN2 is primarily cytoplasmic; assess cytoplasmic staining intensity and distribution

  • Document any unexpected subcellular localization (nuclear, membranous)

  • Note heterogeneity of expression within the sample

• Scoring systems:

  • Use standardized scoring methods (H-score, Allred score, or percentage of positive cells)

  • Establish clear intensity thresholds (negative, weak, moderate, strong)

  • Set validated cutoff values for "high" versus "low" expression based on clinical outcomes

• Technical artifacts to exclude:

  • Edge artifacts (stronger staining at tissue edges)

  • Necrotic tissue staining (often nonspecific)

  • Staining in stromal cells versus tumor cells

  • Background staining due to insufficient blocking

• Clinical correlation challenges:

  • Consider tumor heterogeneity when using tissue microarrays

  • Account for treatment history of the patient

  • Incorporate molecular subtype information when available

  • Correlate with other biomarkers (e.g., mesenchymal markers in GBM)

• Validation approaches:

  • Confirm expression with alternative detection methods (RT-qPCR, Western blot)

  • Compare results across different RHPN2 antibody clones

  • Include appropriate positive and negative control tissues

What are the recommended protocols for detecting RHPN2 interaction with RhoA in experimental systems?

To investigate RHPN2-RhoA interactions, several complementary approaches can be employed:

• RhoA activity assays:

  • RhoA pull-down assay: Use GST-tagged Rhotekin-RBD to selectively capture active RhoA-GTP

  • Sample protocol:

    • Transfect cells with RHPN2 expression vectors or siRNA

    • Serum-starve cells (0.1% FBS, 24h) before stimulation

    • Stimulate with serum (10% FBS, 30min) to activate RhoA

    • Lyse cells and perform pull-down with GST-Rhotekin-RBD

    • Detect active RhoA by Western blot

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of RHPN2 (e.g., FLAG-RHPN2) in appropriate cell lines

  • Immunoprecipitate RHPN2 and probe for co-precipitated RhoA

  • Conversely, immunoprecipitate RhoA and detect associated RHPN2

  • Include controls with RHPN2 mutants defective in Rho binding (e.g., V73M)

• Microscopy-based approaches:

  • Proximity ligation assay (PLA): Detect RHPN2-RhoA interactions in situ within cells

  • FRET analysis: Use fluorescently tagged RHPN2 and RhoA to measure direct interactions

  • Immunofluorescence co-localization: Visualize spatial relationships between RHPN2 and RhoA

• Domain mapping:

  • Generate truncated RHPN2 constructs to identify minimal RhoA-binding domains

  • Create point mutations in key residues to disrupt specific interactions

  • Test binding affinity of wild-type versus mutant RHPN2 to RhoA

When performing these assays, it's critical to include appropriate controls and to consider the specific cell type and experimental conditions that might influence RHPN2-RhoA interactions .

How can RHPN2 expression be effectively manipulated in experimental models to study its function?

Several approaches can be employed to modulate RHPN2 expression for functional studies:

• Overexpression systems:

  • Plasmid-based expression: pcDNA3.1-RHPN2 has been successfully used in multiple studies

  • Viral vectors: Lentiviral or retroviral systems for stable integration

  • Inducible systems: Tet-On/Off systems for temporal control of expression

  • Tagged constructs: FLAG, HA, or GFP-tagged RHPN2 for easy detection and immunoprecipitation

• Knockdown/knockout approaches:

  • siRNA transfection: Transient knockdown (effective for 48-72 hours)

    • Previously validated siRNA sequences are available in published studies

  • shRNA expression: For stable, long-term knockdown

    • Multiple shRNA sequences targeting different regions of RHPN2 have been validated

  • CRISPR/Cas9 genome editing: For complete knockout

    • Design guide RNAs targeting early exons or essential domains

• Domain-specific mutants:

  • Rho-binding domain mutants (e.g., V73M) to disrupt RhoA interaction

  • Phosphorylation site mutants to study post-translational regulation

  • Deletion constructs to identify functional domains

• Model systems:

  • Cell lines with varying baseline RHPN2 expression:

    • High expression: SNB19 glioma cells

    • Low expression: SF188, THLE-2 cells

  • Primary cells: Human astrocytes, neural stem cells

  • Animal models: Consider xenograft models for in vivo studies

When manipulating RHPN2 expression, it's important to verify the effectiveness of your approach using RT-qPCR and Western blot, and to include appropriate controls .

What methodological approaches can be used to study RHPN2's role in cancer cell invasion and migration?

To comprehensively investigate RHPN2's impact on cancer cell invasion and migration, a multi-assay approach is recommended:

• Invasion assays:

  • Transwell Matrigel invasion assay:

    • Coat upper chamber with Matrigel (typically 100-300 μg/ml)

    • Seed cells in serum-free medium in upper chamber

    • Add chemoattractant (10% FBS) to lower chamber

    • Incubate 24-48 hours

    • Fix, stain, and count cells that have invaded through Matrigel

  • 3D spheroid invasion assay:

    • Form tumor spheroids in non-adherent conditions

    • Embed spheroids in 3D matrix (Matrigel, collagen)

    • Monitor invasion radially from spheroid over time

• Migration assays:

  • Wound healing/scratch assay:

    • Create a "wound" in confluent monolayer

    • Measure wound closure over time

    • Consider mitomycin C treatment to inhibit proliferation

  • Single-cell tracking:

    • Track individual cell movements using time-lapse microscopy

    • Analyze velocity, directionality, and persistence

• Cytoskeletal analysis:

  • Immunofluorescence staining:

    • Visualize F-actin organization using phalloidin

    • Examine focal adhesions using anti-paxillin or anti-vinculin antibodies

    • Assess stress fiber formation and cell morphology

  • Live-cell imaging of cytoskeletal dynamics:

    • Use fluorescently labeled actin or LifeAct probes

• Molecular mechanism investigation:

  • RhoA activity assays (as described in FAQ 4.3)

  • Matrix metalloproteinase (MMP) expression and activity analysis

  • Cell-matrix adhesion assays

This comprehensive approach has been successfully employed in studies demonstrating RHPN2's role in promoting invasion and migration in glioblastoma models .

What are the emerging approaches for targeting RHPN2 or its signaling pathways therapeutically?

While RHPN2-targeted therapies are still in early research stages, several promising approaches are being explored:

• Direct targeting of RHPN2:

  • RNA interference: siRNA or antisense oligonucleotides delivery systems

  • Protein-protein interaction inhibitors: Small molecules disrupting RHPN2-RhoA interaction

  • Proteasomal degradation induction: PROTACs (Proteolysis Targeting Chimeras) approach

• Targeting RHPN2-regulated pathways:

  • RhoA pathway inhibitors: ROCK inhibitors (e.g., Y-27632, Fasudil)

  • c-Myc stability modulators: Compounds affecting Ser62 phosphorylation

  • Glutamine metabolism inhibitors: Targeting glutamine synthetase (GS) or glutaminase

• Combination approaches:

  • Sensitization to glutamine deprivation: Combining RHPN2 inhibition with glutaminase inhibitors

  • Cytoskeletal-targeting agents: Pairing with microtubule inhibitors

  • Immune checkpoint inhibitors: Potential synergy with immune therapy

• Biomarker strategies:

  • Using RHPN2 expression as a predictive biomarker for response to specific therapies

  • Stratifying patients based on RHPN2 amplification/overexpression for clinical trials

  • Monitoring RHPN2 levels during treatment to assess response

• Delivery challenges and solutions:

  • Nanoparticle-based delivery systems for RNA therapeutics

  • Blood-brain barrier penetration strategies for glioblastoma treatment

  • Tumor-specific targeting approaches

These approaches are supported by preclinical evidence that RHPN2 inhibition can reduce cancer cell invasion, promote apoptosis, and sensitize cells to therapy in various cancer models .

What are the key unanswered questions regarding RHPN2's role in different cancer types?

Despite progress in understanding RHPN2's functions, several critical questions remain:

• Cancer type specificity:

  • Why does RHPN2 affect proliferation in hepatocellular carcinoma but not in glioblastoma?

  • Are there tissue-specific binding partners that alter RHPN2 function?

  • What determines the contradictory effects on RhoA activity in different cancers?

• Upstream regulation:

  • What signals or pathways regulate RHPN2 expression and activity?

  • Are there post-translational modifications that affect RHPN2 function?

  • How is RHPN2 amplification mechanistically linked to mesenchymal transformation?

• Pathway integration:

  • How does RHPN2 interact with other cancer-associated pathways beyond RhoA?

  • What is the relationship between RHPN2 and other Rho family GTPases?

  • How does RHPN2 influence tumor microenvironment interactions?

• Therapeutic potential:

  • Can RHPN2 be effectively targeted in vivo?

  • Will RHPN2 inhibition have different effects across cancer types?

  • What biomarkers might predict response to RHPN2-targeted therapies?

• Role in cancer progression:

  • Does RHPN2 play roles in early carcinogenesis or primarily in advanced disease?

  • Is RHPN2 involved in metastatic processes beyond local invasion?

  • How does RHPN2 contribute to therapy resistance mechanisms?

Addressing these questions will require integrated approaches combining genomic, proteomic, and functional studies across multiple cancer types .

How might new technologies advance our understanding of RHPN2's functions and interactions?

Emerging technologies offer exciting opportunities to deepen our understanding of RHPN2 biology:

• Single-cell analysis techniques:

  • Single-cell RNA sequencing to identify cell populations with distinct RHPN2 expression patterns

  • Single-cell proteomics to map RHPN2 protein networks at cellular resolution

  • Spatial transcriptomics to evaluate RHPN2 expression in the context of tumor microenvironment

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize RHPN2-cytoskeleton interactions

  • Live-cell imaging combined with optogenetic control of RHPN2 activity

  • Correlative light and electron microscopy to understand RHPN2's subcellular localization

• Proteomic technologies:

  • Proximity labeling (BioID, APEX) to identify novel RHPN2 interaction partners

  • Crosslinking mass spectrometry to map protein-protein interaction interfaces

  • Thermal proteome profiling to discover compounds targeting RHPN2

• Gene editing advancements:

  • CRISPR interference/activation for endogenous gene modulation

  • Base editing and prime editing for precise mutation introduction

  • Inducible CRISPR systems for temporal control of gene editing

• Computational approaches:

  • Machine learning algorithms to predict RHPN2 function from structural data

  • Network analysis of multi-omics data to position RHPN2 in cancer pathways

  • Patient-derived xenograft data integration with genomic information

These technological advances promise to resolve contradictions in the literature and identify new therapeutic opportunities targeting RHPN2 and its downstream pathways .

What considerations should researchers take into account when designing long-term studies of RHPN2 in cancer progression?

Designing robust longitudinal studies of RHPN2 in cancer requires careful consideration of multiple factors:

• Model system selection:

  • Cell lines: Use multiple lines representing different cancer subtypes and RHPN2 expression levels

  • Patient-derived models: Consider PDX models or organoids to maintain tumor heterogeneity

  • Genetic models: Develop conditional RHPN2 transgenic or knockout animals

  • Orthotopic models: Use anatomically appropriate implantation sites

• Temporal considerations:

  • Implement sampling at multiple time points during tumor progression

  • Use inducible systems to activate or suppress RHPN2 at defined stages

  • Consider aging effects in long-term animal studies

• Heterogeneity assessment:

  • Analyze multiple regions within tumors

  • Use single-cell approaches to resolve subpopulation differences

  • Track clonal evolution of RHPN2-altered cells

• Comprehensive phenotyping:

  • Assess multiple hallmarks of cancer beyond invasion (metabolism, immune evasion)

  • Measure treatment response longitudinally

  • Evaluate distant metastasis, not just local invasion

• Translational relevance:

  • Correlate findings with patient samples at different disease stages

  • Develop clinically relevant biomarkers for RHPN2 activity

  • Consider standard-of-care treatments in experimental design

• Reproducibility and validation:

  • Use multiple technical approaches for key findings

  • Validate across different model systems

  • Implement rigorous statistical planning for longitudinal analyses