STARD8 Antibody

What is STARD8 and why is it significant in cancer research?

STARD8, also known as DLC3 (deleted in liver cancer protein 3), is a Rho-GTPase-activating protein that functions as a tumor suppressor gene. It maps to chromosome Xq13 and was first isolated from a human myeloid cell line library in 1996 . STARD8 is significant in cancer research because:

• It shows downregulation in multiple cancer types including gastric, breast, ovarian, liver, and prostate cancers

• Its decreased expression significantly correlates with TNM stage, lymph node metastasis, and differentiation in gastric cancer

• Overexpression of STARD8 in cancer cell lines represses cell proliferation and colony formation, suggesting a tumor suppressor role

• It may be involved in regulating cell morphology through its effects on the cytoskeleton

Research using STARD8 antibodies helps in understanding its expression patterns, localization, and role in various cancers, potentially identifying new therapeutic targets or prognostic markers.

What are the structural domains of STARD8 and how do they relate to its function?

STARD8 contains three principal functional domains that contribute to its tumor suppressor activity:

• Sterile alpha motif (SAM) domain: Located at the N-terminus, involved in protein-protein interactions

• RhoGAP domain: Central domain responsible for GTPase-activating function

• START (steroidogenic acute regulatory protein-related lipid transfer) domain: C-terminal domain involved in lipid binding and transfer

This multi-domain structure enables STARD8 to:

• Accelerate GTPase activity specifically for RHOA and CDC42, but not RAC1

• Stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLCD1

• Localize to focal adhesions and co-localize with vinculin

• Regulate cell morphology and potentially inhibit cancer cell migration and invasion

Understanding these domains helps researchers select antibodies targeting specific regions for different experimental applications.

How should I select the appropriate STARD8 antibody for my research?

When selecting a STARD8 antibody, consider the following criteria based on your experimental needs:

For immunohistochemistry applications, antibodies validated with appropriate antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) are preferable .

How can I validate the specificity of a STARD8 antibody?

Proper validation ensures reliable experimental results. Implement these strategies to confirm STARD8 antibody specificity:

• Western blot analysis: Confirm a single band of appropriate molecular weight (113-120 kDa)

• Positive controls: Use cell lines known to express STARD8, such as GES1 (normal gastric mucosa cell line)

• Negative controls: Include samples with low or no STARD8 expression, such as certain gastric cancer cell lines like HGC27

• siRNA knockdown: Use STARD8-specific siRNA (such as StARD8 siRNA sc-63080) to create negative controls through knockdown

• Multiple detection methods: Compare results across different techniques (WB, IHC, IF, RT-PCR)

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm binding specificity

As noted in search result , comprehensive validation may include comparing mRNA expression (by RT-PCR) with protein expression (by Western blot and immunohistochemistry) across multiple cell lines or tissue samples.

What are the recommended dilutions and protocols for STARD8 antibodies in different applications?

The optimal dilutions vary by specific antibody and application:

For IHC scoring, the protocol in recommends a semi-quantitative analysis based on extent (percentage of positive cells) and intensity of staining:

• Extent scoring: 0≤5%, 1=6%–25%, 2=26%–50%, 3=51%–75%, 4≥75%

• Intensity scoring: 0=achromatic, 1=light yellow, 2=yellow, 3=brown

• Combined score: Multiply extent by intensity [(−)=0, (+)=1–4, (++)=5–8, (+++)=9–12]

How can I optimize STARD8 detection in immunofluorescence studies?

To achieve optimal immunofluorescence visualization of STARD8:

• Cell preparation: Culture cells to confluence on collagen-coated glass coverslips

• Fixation: Use 4% paraformaldehyde in 50% Tris wash buffer (TWB)

• Permeabilization: Treat with 1.2% Triton X-100 for 10 minutes

• Blocking: Incubate with 5% bovine serum albumin (BSA) in 100% TWB for 2 hours

• Primary antibody: Incubate with mouse polyclonal antibody against human STARD8 at 1:100 dilution

• Secondary antibody: Use appropriate fluorophore-conjugated secondary antibodies (e.g., rabbit anti-mouse IgG conjugated with Tritic)

• Counterstaining: Include nuclear counterstain (DAPI) for localization reference

• Mounting: Use anti-fade mounting medium to prevent photobleaching

Pay special attention to subcellular localization, as STARD8 is present in both nucleus and cytoplasm, with stronger nuclear localization in normal cells .

What are common issues encountered with STARD8 antibodies in Western blotting?

For best results, use freshly prepared cell lysates, include appropriate controls, and optimize blocking and antibody incubation conditions for your specific experimental system.

How can I address variable staining patterns in STARD8 immunohistochemistry?

Variable staining patterns may result from several factors:

• Tissue fixation variations: Standardize fixation protocols (10% neutral buffered formalin recommended)

• Antigen retrieval differences: Compare TE buffer pH 9.0 (recommended) with citrate buffer pH 6.0

• Antibody selection: Different antibodies may recognize different epitopes or isoforms

• Cancer heterogeneity: STARD8 expression varies with differentiation status and cancer progression

• Technical variations: Standardize incubation times, temperatures, and detection systems

The semiquantitative scoring system described in can help normalize these variations:

• Two independent assessors should perform scoring without prior knowledge of patient outcome

• Low expression = scores (−) or (+); High expression = scores (++) or (+++)

• Correlate expression with clinicopathological parameters for meaningful interpretation

How can STARD8 antibodies be used to investigate its role in tumor suppression?

STARD8 antibodies enable several approaches to study its tumor suppressor function:

• Expression profiling across cancer types:

  • Compare STARD8 levels between normal and cancer tissues using IHC and Western blot

  • Correlate expression with tumor grade, stage, and patient outcomes

• Mechanistic studies:

  • Investigate interactions with Rho GTPases through co-immunoprecipitation and co-localization studies

  • Examine effects on focal adhesion dynamics through immunofluorescence with focal adhesion markers

• Functional validation:

  • After STARD8 overexpression or knockdown, assess:

    • Cell proliferation and colony formation

    • Migration and invasion capabilities

    • Cell morphology changes and cytoskeletal reorganization

• Biomarker development:

  • Evaluate STARD8 as a prognostic biomarker through large-scale IHC studies

  • Correlate with clinical parameters such as TNM stage, lymph node metastasis, and differentiation

Research in gastric cancer has demonstrated that STARD8 expression is inversely correlated with differentiation, TNM type, and lymph node metastasis (p<0.05) , supporting its role as a tumor suppressor.

What approaches can reveal STARD8's relationship with Rho GTPase signaling?

To investigate STARD8's RhoGAP activity and interactions with Rho GTPases:

• Protein-protein interaction studies:

  • Co-immunoprecipitation using STARD8 antibodies to pull down associated Rho GTPases

  • Proximity ligation assay (PLA) to visualize in situ interactions

  • GST pull-down assays with GTP-loaded RhoA and Cdc42

• Functional GTPase assays:

  • Measure GTP hydrolysis of RhoA and Cdc42 in the presence of immunoprecipitated STARD8

  • Use phospho-specific antibodies against Rho GTPase targets to assess pathway activation

• Localization studies:

  • Co-immunofluorescence of STARD8 with RhoA and Cdc42

  • Visualization of active Rho GTPases using specific biosensors in cells with modulated STARD8 expression

• Downstream effector analysis:

  • Examine how STARD8 affects actin cytoskeleton organization

  • Assess focal adhesion dynamics through co-localization with vinculin

These approaches can help define the molecular mechanisms underlying STARD8's tumor suppressor function through its regulation of Rho GTPase signaling.

How should STARD8 expression data be quantified and interpreted in cancer studies?

For rigorous quantification and interpretation of STARD8 expression:

• For immunohistochemistry:

  • Use the validated scoring system from :

    • Extent score: 0≤5%, 1=6%–25%, 2=26%–50%, 3=51%–75%, 4≥75%

    • Intensity score: 0=achromatic, 1=light yellow, 2=yellow, 3=brown

    • Combined score: Extent × Intensity, categorized as (−)=0, (+)=1–4, (++)=5–8, (+++)=9–12

  • Have two independent assessors evaluate samples blindly

  • Compare expression between tumor and adjacent normal tissue from the same patient

• For Western blot:

  • Normalize to housekeeping proteins (β-actin recommended)

  • Perform densitometric analysis across multiple samples

  • Express results as fold-change relative to appropriate controls

• For RT-PCR:

  • Use appropriate primers (see Table 1 from search result for STARD8 primers)

  • Normalize to housekeeping genes (GAPDH recommended)

  • Calculate relative expression using comparative Ct method

• Statistical analysis:

  • Correlate expression with clinicopathological features using appropriate statistical tests

  • Consider multivariate analysis to identify independent prognostic factors

In gastric cancer studies, low STARD8 expression correlated significantly with poorer differentiation, advanced TNM stage, and presence of lymph node metastasis .

What are the implications of different subcellular localization patterns of STARD8?

The subcellular distribution of STARD8 provides important functional insights:

• Normal localization pattern:

  • STARD8 localizes to both nucleus and cytoplasm, but more strongly to the nucleus in normal cells

  • Also found at focal adhesions where it co-localizes with vinculin

• Interpreting localization changes:

  • Altered nuclear/cytoplasmic ratio may indicate functional changes

  • Loss of nuclear localization might suggest impaired tumor suppressor function

  • Changes in focal adhesion localization may affect cell adhesion and migration

• Functional implications:

  • Nuclear STARD8 may be involved in transcriptional regulation

  • Cytoplasmic STARD8 likely participates in Rho GTPase regulation

  • Focal adhesion-associated STARD8 regulates cell morphology and migration

• Technical considerations:

  • Use confocal microscopy for precise subcellular localization assessment

  • Perform nuclear/cytoplasmic fractionation followed by Western blotting for quantitative analysis

  • Include co-staining with appropriate subcellular markers (nuclear, cytoplasmic, focal adhesion)

How might STARD8 antibodies be used in developing potential cancer biomarkers?

STARD8 antibodies show promise for biomarker development in several ways:

• Prognostic biomarker development:

  • Large-scale IHC studies correlating STARD8 expression with patient outcomes

  • Multi-cancer analysis to determine cancer type-specific expression patterns

  • Integration with other biomarkers for improved prognostic power

• Predictive biomarker applications:

  • Assess whether STARD8 expression levels predict response to specific therapies

  • Determine if STARD8 status correlates with sensitivity to cytoskeletal-targeting drugs

  • Investigate potential synthetic lethality approaches based on STARD8 status

• Methodological approaches:

  • Tissue microarray analysis for high-throughput screening

  • Automated image analysis for standardized quantification

  • Multiplex IHC to assess STARD8 alongside other pathway components

• Clinical translation considerations:

  • Standardization of antibodies and protocols for clinical use

  • Establishment of clinically relevant cutoff values

  • Validation in prospective clinical trials

Early research suggests that low STARD8 expression correlates with poor differentiation, advanced TNM stage, and lymph node metastasis in gastric cancer , indicating potential utility as a prognostic biomarker.

What new methodologies might enhance STARD8 protein research?

Emerging technologies that could advance STARD8 research include:

• Advanced imaging approaches:

  • Super-resolution microscopy for detailed focal adhesion localization

  • Live-cell imaging with tagged STARD8 to monitor dynamic localization

  • FRET-based biosensors to assess STARD8-Rho GTPase interactions in real-time

• Proteomic technologies:

  • Proximity-dependent biotinylation (BioID or TurboID) to identify STARD8 interactors

  • Phosphoproteomics to characterize STARD8 phosphorylation sites and signaling

  • Protein array technologies for high-throughput interaction studies

• Genetic engineering approaches:

  • CRISPR/Cas9 gene editing to create cellular models with tagged endogenous STARD8

  • Domain-specific mutations to dissect functions of individual STARD8 domains

  • Inducible expression systems to study acute effects of STARD8 modulation

• Single-cell technologies:

  • Single-cell protein analysis to assess STARD8 heterogeneity in tumors

  • Spatial transcriptomics combined with STARD8 IHC for contextual expression analysis

  • Multiparameter analysis correlating STARD8 with cell state markers

These advanced methodologies could significantly enhance our understanding of STARD8's role in normal physiology and cancer progression, potentially revealing new therapeutic opportunities.

What are the critical considerations when planning STARD8 antibody-based experiments?

When designing STARD8 antibody experiments, researchers should consider:

Careful consideration of these factors will enhance the reliability and reproducibility of STARD8 antibody-based research.

What unresolved questions about STARD8 might antibody-based research help address?

Several important questions about STARD8 remain unanswered and could be investigated using antibody-based approaches:

• Regulation mechanisms:

  • How is STARD8 expression and localization regulated in normal versus cancer cells?

  • What post-translational modifications affect STARD8 function?

  • How do tumor microenvironment factors influence STARD8 expression?

• Functional interplay:

  • How does STARD8 coordinate with other tumor suppressors like DLC1 and DLC2?

  • What is the complete interactome of STARD8 beyond Rho GTPases?

  • How do the three domains (SAM, RhoGAP, START) cooperate functionally?

• Clinical significance:

  • Does STARD8 downregulation occur in additional cancer types beyond those already studied?

  • Can STARD8 status predict therapeutic response to specific targeted therapies?

  • Is there potential to develop therapeutics that restore STARD8 function?

• Mechanistic details:

  • What are the precise molecular mechanisms by which STARD8 regulates focal adhesion dynamics?

  • How does nuclear STARD8 contribute to its tumor suppressor function?

  • What role does the START domain play in STARD8's tumor suppressor activity?