RASSF7 Antibody

What is RASSF7 and what is its biological significance in cells?

RASSF7 (Ras association domain-containing protein 7), previously known as HRC1 (HRAS1 cluster 1) and C11orf13, is a 373 amino acid protein that belongs to the N-terminal RASSF family. This evolutionary conserved group includes RASSF7, RASSF8, RASSF9 (P-CIP1), and RASSF10 . RASSF7 is broadly expressed across different tissues and cell types, with particularly high expression observed in the lung and brain .

Unlike the classical RASSF proteins (RASSF1-RASSF6), which are generally considered tumor suppressors, RASSF7 expression is actually upregulated in various cancers . RASSF7 plays essential roles in:

• Mitotic regulation, particularly spindle formation and chromosomal congression

• Microtubule dynamics and growth from the centrosome

• Negative regulation of stress-induced JNK activation and apoptosis

• Promotion of MAP2K7 phosphorylation

RASSF7 is critical for normal cell division, as knockdown studies show severe mitotic defects leading to cell growth inhibition .

Where is RASSF7 protein localized in cells and how is this determined?

RASSF7 protein predominantly localizes to the centrosome, a finding consistently demonstrated across multiple studies and species . The centrosomal localization has been determined through:

• Immunofluorescence staining with anti-RASSF7 antibodies showing co-localization with γ-tubulin (a component of the pericentriolar material)

• GFP-tagged RASSF7 protein expression studies

• Analyses in both human and Xenopus cells

Importantly, RASSF7 localizes to the centrosome throughout the cell cycle - in both interphase and all stages of mitosis - indicating it is not specifically recruited during mitosis but is a constitutive centrosomal component . This localization is microtubule-independent in human cells, as demonstrated by nocodazole treatment experiments which showed that RASSF7 remains at the centrosome even after microtubule depolymerization .

How does RASSF7 influence the activation of Aurora B during mitosis?

RASSF7 is required for proper Aurora B activation at the kinetochores during mitosis, though not directly at the centrosome where RASSF7 is localized. Multiple lines of evidence support this:

• In RASSF7-knockdown cells, phospho-specific antibodies detecting active Aurora kinases showed strongly reduced staining at the kinetochores (only 9.3% of cells showing strong Aurora B staining compared with 83.8% in controls) .

• The phosphorylation of CENP-A, a direct target of Aurora B, was significantly reduced in RASSF7-knockdown cells (only 22% of cells showing strong staining compared with 93.4% of control cells) .

• The localization of Aurora B itself at the kinetochores remained intact in RASSF7-knockdown cells, indicating the issue is with activation rather than recruitment .

• Interestingly, Aurora B activation during cytokinesis appeared normal in RASSF7-knockdown cells, suggesting the requirement for RASSF7 is specific to metaphase Aurora B activation .

The mechanism linking centrosomal RASSF7 to kinetochore Aurora B activation likely involves microtubule dynamics, as Aurora B activation requires contact with microtubules. The defective spindle formation in RASSF7-knockdown cells likely prevents proper microtubule-Aurora B interaction, explaining this phenotype .

What experimental approaches should be used to study RASSF7's role in microtubule dynamics?

To effectively study RASSF7's influence on microtubule dynamics, researchers should consider the following experimental approaches:

Microtubule regrowth assays:

• Treat cells with nocodazole to depolymerize microtubules

• Wash out the drug and examine microtubule regrowth at specific time points (5, 15, 30, and 60 minutes are recommended)

• Compare control and RASSF7-knockdown cells, focusing on both the rate of regrowth and microtubule morphology

Spindle formation analysis:

• Use immunofluorescence microscopy to visualize mitotic spindles in RASSF7-knockdown cells

• Specifically assess spindle polarization, microtubule organization, and the frequency of multi-polar spindles

Live cell imaging:
Since RASSF7 impacts dynamic processes, time-lapse microscopy of fluorescently tagged microtubules in control and RASSF7-depleted cells can provide valuable insights into the temporal aspects of microtubule dynamics.

Biochemical interaction studies:
Investigate whether RASSF7 directly interacts with tubulin or microtubule-associated proteins through co-immunoprecipitation and in vitro binding assays.

These approaches should be combined with proper controls, including rescue experiments where wild-type RASSF7 is reintroduced into knockdown cells to confirm specificity of the observed phenotypes.

How does RASSF7 expression compare between normal and cancer tissues, and what are the implications?

RASSF7 shows intriguing expression patterns in cancer contexts that differ from most RASSF family members:

This expression pattern contrasts with other RASSF family members:

• RASSF8 shows reduced expression in lung cancers

• RASSF10 expression is epigenetically inactivated in leukemias and thyroid cancers

• RASSF1-6 generally function as tumor suppressors

The implications of increased RASSF7 expression in cancer are significant:

• RASSF7 may not function as a tumor suppressor like other family members

• RASSF7 could potentially promote cancer formation

• The hypoxic environment in solid tumors likely explains the increased RASSF7 expression observed

• RASSF7 may be required for the growth of cancer cells, as knockdown inhibits anchorage-independent growth

These findings suggest RASSF7 could potentially serve as a therapeutic target similar to PLK1, where inhibition causes mitotic arrest and cell death .

What are the consequences of RASSF7 knockdown in different experimental systems?

RASSF7 knockdown produces consistent and severe phenotypes across different experimental systems:

In human cell lines (HeLa and H1792):

• Reduced cell number and inhibited anchorage-independent growth

• Mitotic aberrations in metaphase cells:

  • 63% of cells failed to align chromosomes (vs. 18% in controls)

  • 21.5% of metaphase cells showed lagging chromosomes (vs. 6% in controls)

  • Only 15.7% of cells displayed correctly aligned DNA (vs. 75.7% in controls)

• Spindle defects:

  • Less pronounced polarization toward DNA

  • More radial organization of microtubules

  • Increased multi-polar spindles (32.6% vs. 23.7% in controls)

• Failure in Aurora B activation at kinetochores

• Delayed and abnormal microtubule regrowth after nocodazole treatment

In Xenopus embryos:

• Failure to form mitotic spindles

• Nuclear fragmentation

• Apoptosis

• Loss of tissue architecture in the neural tube

Interestingly, the increase in apoptosis observed in Xenopus was not detected in HeLa cells, where the percentage of active caspase 3-positive cells was not significantly different between RASSF7-knockdown and control cells . This suggests some context-dependent differences in the downstream consequences of RASSF7 depletion.

What are the optimal conditions for using RASSF7 antibodies in immunofluorescence studies?

Based on published research protocols, the following conditions are recommended for optimal RASSF7 immunofluorescence staining:

For HeLa cells:

• Fix cells in 4% formaldehyde

• Permeabilize using 0.2% Triton X-100

• Block in 10% normal goat serum

• Incubate with anti-RASSF7 antibody (recommend 1:100-1:200 dilution)

• Detect using appropriate secondary antibody (e.g., Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG)

• Co-stain with γ-tubulin antibody to confirm centrosomal localization

For HepG2 cells:

• Fix cells in 4% formaldehyde

• Permeabilize using 0.2% Triton X-100

• Use anti-RASSF7 antibody at 1/133 dilution

• Detect with Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG secondary antibody

Important considerations:

• RASSF7 shows strong centrosomal staining that co-localizes with γ-tubulin

• Include appropriate negative controls, such as RASSF7-knockdown cells

• For mitotic studies, co-stain with DNA markers to identify mitotic stages

• Consider adding microtubule markers (α-tubulin) to analyze spindle formation simultaneously

How can researchers effectively differentiate between RASSF7 and other RASSF family members?

Distinguishing RASSF7 from other RASSF family members is crucial for accurate research. Consider these approaches:

Antibody selection:

• Use RASSF7-specific antibodies validated for specificity with proper controls

• Confirm antibody specificity by testing against RASSF7-knockdown samples

• Verify the antibody recognizes the expected molecular weight (34-40 kDa)

Structural and functional differences:

• RASSF7 belongs to the N-terminal RASSF family (RASSF7-10), which is structurally distinct from classical RASSF proteins (RASSF1-6)

• RASSF7 localizes to the centrosome, which differs from the localization of many other RASSF proteins

• RASSF7 is required for growth, unlike RASSF6 and RASSF8, which restrain growth

• Expression patterns differ: RASSF7 is upregulated in some cancers, while most RASSF proteins show reduced expression

Experimental validation:

• Perform siRNA knockdown with RASSF7-specific sequences to confirm phenotypes are due to RASSF7 rather than other family members

• Use at least two different siRNA/shRNA sequences to ensure specificity

• Include rescue experiments with siRNA-resistant RASSF7 constructs to confirm specificity

What controls should be included when studying RASSF7 in mitosis experiments?

When investigating RASSF7's role in mitosis, include these essential controls:

For knockdown studies:

• Non-targeting siRNA/shRNA control

• Multiple different RASSF7-targeting sequences to rule out off-target effects

• Rescue experiments with siRNA/shRNA-resistant RASSF7 constructs

• Western blot verification of knockdown efficiency

For immunofluorescence:

• RASSF7-knockdown cells as negative control for antibody specificity

• Co-staining with centrosomal markers (γ-tubulin) to confirm localization

• Include mitotic markers to properly identify cell cycle stages

• For Aurora B activation studies, include controls for both localization (INCENP) and activity (phospho-CENP-A)

For functional assays:

• Positive controls for mitotic defects (e.g., Aurora B inhibitor)

• Time-course experiments for dynamic processes like microtubule regrowth

• Multiple time points during mitosis to capture the full sequence of events

For expression studies:

• Multiple cell lines to ensure findings are not cell-type specific

• Hypoxia controls when studying cancer contexts, as RASSF7 is hypoxia-responsive

What are the technical challenges in detecting endogenous RASSF7 protein, and how can they be overcome?

Researchers face several challenges when detecting endogenous RASSF7:

Challenge 1: Low basal expression levels

• Solution: Enhance detection sensitivity using signal amplification techniques

• Solution: Consider studying RASSF7 in hypoxic conditions where its expression is naturally increased

Challenge 2: Multiple isoforms

• RASSF7 has three isoforms with molecular weights of 40 kDa, 36 kDa, and 35 kDa

• Solution: Use antibodies that recognize a common epitope across all isoforms

• Solution: Clearly document which isoform(s) are being detected in your experimental system

Challenge 3: Centrosomal localization
The centrosome is a small organelle, making visualization challenging.

• Solution: Use high-resolution microscopy techniques

• Solution: Co-stain with established centrosomal markers like γ-tubulin

• Solution: Consider structured illumination or confocal microscopy for better resolution

Challenge 4: Antibody specificity

• Solution: Validate antibodies using RASSF7-knockdown cells as negative controls

• Solution: Compare multiple commercially available antibodies

• Solution: Consider using tagged RASSF7 constructs as positive controls

Challenge 5: Cell cycle-dependent effects

• Solution: Synchronize cells for studying specific cell cycle stages

• Solution: Use cell cycle markers to clearly identify mitotic versus interphase cells

How might RASSF7 function as a potential therapeutic target in cancer research?

RASSF7 presents several characteristics that make it a potential therapeutic target:

• Upregulation in cancer: Unlike many tumor suppressors, RASSF7 expression is increased in various cancers, including an 87-fold upregulation in pancreatic islet cell tumors . This suggests it may play a pro-oncogenic role.

• Essential for growth: Knockdown of RASSF7 inhibits anchorage-independent growth and reduces cell numbers, indicating cancer cells may depend on RASSF7 for survival and proliferation .

• Mitotic function: Inhibiting RASSF7 causes arrest in mitosis followed by cell death, similar to the effect of PLK1 inhibition . PLK1 inhibitors are currently being developed for clinical use, suggesting a parallel approach could be taken for RASSF7 .

• Centrosomal localization: As a centrosome-associated protein, RASSF7 may be targetable with approaches that disrupt this localization, potentially affecting cancer cells more than normal cells due to their heightened mitotic activity.

• Hypoxia responsiveness: The increased expression of RASSF7 under hypoxic conditions, which are common in solid tumors, provides a potential window of therapeutic selectivity .

Future research directions should include:

• High-throughput screening for small molecule inhibitors of RASSF7

• Investigation of RASSF7's protein interaction network to identify additional druggable nodes

• Evaluation of synthetic lethality approaches in RASSF7-overexpressing tumors

• Development of strategies to disrupt RASSF7's centrosomal localization

What cell models and experimental systems are most suitable for studying RASSF7 function?

Based on the available research, these models and systems have proven effective for RASSF7 studies:

Cell Lines Successfully Used:

• HeLa cells: Well-characterized for mitosis studies and RASSF7 knockdown experiments

• H1792 cells: Human lung adenocarcinoma line used for anchorage-independent growth assays

• HepG2 cells: Human liver hepatocellular carcinoma cells shown to express RASSF7

• HL-60 cells: Human promyelocytic leukemia cells verified to express RASSF7

• PC-12 cells: Rat pheochromocytoma cell line verified to express RASSF7

Animal Models:

• Xenopus embryos: Successfully used for developmental studies of RASSF7

• Mouse embryos: Used for in situ hybridization to study tissue expression patterns

Experimental Systems:

• RNAi knockdown: Both siRNA and shRNA approaches have been successful

• GFP-tagged RASSF7: Used for localization studies

• Nocodazole-based microtubule regrowth assays: Effective for studying RASSF7's role in microtubule dynamics

• Soft agar colony formation assays: Used to assess RASSF7's role in anchorage-independent growth

When selecting a model system, consider:

• Expression level of endogenous RASSF7 in the cell type

• Research question (mitosis, microtubule dynamics, or hypoxia response)

• Technical requirements (transfection efficiency, imaging accessibility)

• Relevance to cancer biology if studying RASSF7 in oncology contexts

How can researchers investigate the relationship between RASSF7 and hypoxia in tumor microenvironments?

To investigate RASSF7-hypoxia relationships in tumor contexts, consider these methodological approaches:

In vitro hypoxia models:

• Culture cells in hypoxic chambers (1-2% O₂) to mimic tumor microenvironments

• Use chemical hypoxia mimetics (e.g., CoCl₂, DMOG) as alternative approaches

• Monitor RASSF7 protein levels via Western blotting at various time points during hypoxia

• Compare RASSF7 mRNA and protein levels to establish regulatory mechanisms

• Perform RASSF7 knockdown under hypoxic conditions to determine functional importance

3D culture systems:

• Develop spheroid models that naturally generate hypoxic cores

• Assess RASSF7 expression gradients from outer (normoxic) to inner (hypoxic) regions

• Apply hypoxia markers (e.g., pimonidazole, HIF-1α staining) alongside RASSF7 detection

Patient-derived samples:

• Analyze RASSF7 expression in tumor sections

• Correlate with hypoxia markers (CA9, GLUT1, HIF-1α)

• Assess relationship with tumor progression and patient outcomes

Mechanistic studies:

• Determine if RASSF7 is directly regulated by HIFs using ChIP assays

• Investigate whether hypoxia-induced RASSF7 expression affects mitotic progression

• Explore if RASSF7 contributes to hypoxia-induced treatment resistance

• Assess if targeting RASSF7 in hypoxic tumor regions provides therapeutic benefit

Key experimental controls:

• Include matched normoxic controls for all hypoxia experiments

• Verify hypoxic conditions using established markers

• Use multiple cell lines to determine consistent patterns

• Include time-course experiments to capture dynamic responses

These approaches will help elucidate how RASSF7 upregulation in hypoxic tumor microenvironments contributes to cancer biology and potentially identify novel therapeutic strategies targeting this relationship.

What strategies can resolve non-specific binding when using RASSF7 antibodies in immunoblotting?

When encountering non-specific binding with RASSF7 antibodies in Western blotting, implement these troubleshooting strategies:

Optimize blocking conditions:

• Extend blocking time (2-3 hours at room temperature or overnight at 4°C)

• Test different blocking agents (5% non-fat milk, 5% BSA, commercial blocking buffers)

• For polyclonal antibodies, consider adding 1-5% serum from the same species as the secondary antibody

Antibody dilution and incubation:

• Test a range of primary antibody dilutions (recommended ranges for RASSF7: 1:500-1:2000 or 1:500-1:1000 )

• Extend primary antibody incubation time at 4°C (overnight)

• Add 0.05-0.1% Tween-20 to antibody dilution buffer

Stringent washing:

• Increase number of washes (5-6 times for 5-10 minutes each)

• Use TBS-T with higher Tween-20 concentration (0.1-0.3%)

Sample preparation:

• Include protease inhibitors in lysis buffer to prevent degradation

• Denature samples thoroughly (95°C for 5 minutes)

• Use fresh samples when possible

Specificity controls:

• Include RASSF7-knockdown cell lysate as negative control

• Note that RASSF7 has three isoforms (40 kDa, 36 kDa, and 35 kDa)

• Major band should migrate at the predicted size (34-40 kDa)

Membrane considerations:

• Use PVDF membranes for potentially better signal-to-noise ratio

• Consider increasing transfer time for higher molecular weight proteins

Alternative antibody:

• Try antibodies targeting different epitopes of RASSF7

• Compare monoclonal vs. polyclonal antibodies

Implementing these strategies systematically should help resolve most non-specific binding issues when detecting RASSF7 by Western blotting.

How can researchers address variability in RASSF7 knockdown efficiency across different experimental systems?

Variability in RASSF7 knockdown efficiency is a common challenge. Here are strategies to address this issue:

siRNA/shRNA optimization:

• Test multiple siRNA/shRNA sequences targeting different regions of RASSF7 mRNA

• Use validated sequences from published studies

• Optimize transfection conditions (reagent concentration, cell density, timing)

• Consider chemically modified siRNAs for improved stability

• For difficult cell types, use electroporation or nucleofection rather than lipid-based transfection

Delivery method selection:

• For transient knockdown: siRNA transfection (effective for 3-5 days)

• For stable knockdown: shRNA in lentiviral vectors

• For hard-to-transfect cells: viral delivery systems

Knockdown verification:

• Verify knockdown at both mRNA (qRT-PCR) and protein (Western blot) levels

• Establish time course of knockdown to determine optimal time window for experiments

• Include appropriate controls (non-targeting siRNA/shRNA)

Cell type considerations:

• Adjust cell density based on growth rate and transfection efficiency

• For cells with high RASSF7 expression, consider double transfection protocol

• Be aware that hypoxia increases RASSF7 expression, which might counteract knockdown

Alternative approaches:

• For complete knockout: Consider CRISPR-Cas9 genome editing

• For inducible systems: Use tetracycline-regulated shRNA expression

• For rescue experiments: Introduce siRNA-resistant RASSF7 constructs

Standardization practices:

• Establish minimum knockdown threshold for experiments (e.g., >70% reduction)

• Normalize functional readouts to the actual knockdown efficiency achieved

• Pool data only from experiments with comparable knockdown levels

By implementing these strategies, researchers can achieve more consistent RASSF7 knockdown and generate more reliable experimental data across different systems.

What is known about the role of RASSF7 in non-mitotic cellular processes?

While RASSF7's mitotic functions are well-documented, emerging evidence suggests important roles in non-mitotic processes:

Stress response and apoptosis regulation:

• RASSF7 negatively regulates stress-induced JNK activation

• It promotes MAP2K7 phosphorylation, inhibiting MAP2K7's ability to activate JNK

• This creates an anti-apoptotic effect under normal conditions

• During prolonged stress, RASSF7 is degraded via the ubiquitin-proteasome pathway, allowing the apoptotic response to proceed

Microtubule regulation in interphase:

• RASSF7 localizes to centrosomes throughout the cell cycle, not just during mitosis

• Microtubule regrowth assays show RASSF7 affects microtubule dynamics in interphase cells

• This suggests potential roles in interphase microtubule organization, cellular transport, or cell shape maintenance

Developmental functions:

• RASSF7 is expressed in multiple embryonic tissues including skin, neural tube, and eye

• Expression varies across adult tissues, suggesting tissue-specific functions

• The neural tube defects in Xenopus embryos after RASSF7 knockdown suggest roles beyond just cell division

Hypoxia response:

• RASSF7 is upregulated at both mRNA and protein levels under hypoxic conditions

• This may connect RASSF7 to cellular adaptations to low oxygen environments

• Potential roles in hypoxia-induced cellular processes beyond proliferation remain to be explored

These non-mitotic functions represent emerging areas for RASSF7 research that may reveal new therapeutic opportunities and biological insights.

How might RASSF7 interact with other centrosomal proteins to regulate microtubule dynamics?

RASSF7's centrosomal localization suggests potential interactions with key centrosomal proteins to regulate microtubule dynamics. While specific interaction partners are still being elucidated, several possibilities exist:

Potential interaction with γ-tubulin ring complex (γ-TuRC):

• RASSF7 co-localizes with γ-tubulin at the centrosome

• γ-TuRC is essential for microtubule nucleation from the centrosome

• RASSF7 could potentially regulate γ-TuRC activity or localization

• This would explain the defects in microtubule regrowth observed in RASSF7-knockdown cells

Aurora A regulation:

PLK1 pathway interactions:

Centrosomal structural proteins:

• RASSF7 could interact with structural components of the pericentriolar material

• This might stabilize the centrosome and provide a platform for microtubule nucleation

Microtubule-associated proteins (MAPs):

• The abnormal microtubule morphology in RASSF7-knockdown cells suggests possible interactions with MAPs

• RASSF7 might regulate MAPs that control microtubule stability or dynamics

Future research directions should include:

• Proteomic analysis of RASSF7 interactome at the centrosome

• Structure-function studies to identify domains required for centrosomal localization and protein interactions

• Super-resolution microscopy to more precisely localize RASSF7 within the centrosome structure

• In vitro reconstitution experiments to test direct effects on microtubule dynamics