VGLL4 Antibody

What is VGLL4 and what are its key functions in cellular biology?

VGLL4 is a transcription cofactor that acts as a specific coactivator for mammalian TEFs (transcription enhancer factors). With a molecular weight of approximately 31 kDa, VGLL4 contains two Tondu (TDU) domains that are critical for its function .

VGLL4 has several important biological functions:

• Tumor suppression: VGLL4 inhibits YAP-induced cell proliferation and tumorigenesis by competing with YAP for binding to TEADs, the core components of the Hippo pathway .

• Immune regulation: VGLL4 regulates tumor PD-L1 expression and immune surveillance. It interacts with IRF2BP2 and promotes its protein stability through inhibiting proteasome-mediated protein degradation .

• Transcriptional regulation: VGLL4 can trigger TEAD4 condensation by inducing its oligomerization, leading to DNA aggregation and transcriptional repression .

What applications are VGLL4 antibodies commonly used for in research?

VGLL4 antibodies are utilized in multiple research applications, including:

• Western Blot (WB): For detecting VGLL4 protein expression levels in cell and tissue lysates .

• Immunohistochemistry (IHC): For examining VGLL4 localization and expression patterns in tissue sections .

• Immunofluorescence (IF): For subcellular localization studies, particularly to differentiate between nuclear and cytoplasmic distribution .

• ELISA: For quantitative detection of VGLL4 protein levels .

• Immunoprecipitation: For studying protein-protein interactions, particularly with TEADs and IRF2BP2 .

Different applications require specific antibody characteristics, so researchers should select antibodies validated for their particular experimental approach.

What is the normal expression pattern of VGLL4 in healthy tissues?

VGLL4 is widely expressed across many tissue types. According to immunohistochemistry studies on normal human tissues:

• Normal lung tissue demonstrates high nuclear VGLL4 expression, with 92.6% (25 out of 27) of samples showing strong nuclear staining .

• VGLL4 demonstrates predominantly nuclear localization in normal tissues, but a more diffused cytoplasmic pattern in cancer tissues, particularly lung adenocarcinomas .

• VGLL4 is also expressed in heart tissue, where it was initially characterized as a regulator of TEF-1-dependent promoters .

Understanding this normal expression pattern is critical for comparative studies with diseased tissues.

How should researchers optimize immunohistochemistry protocols for VGLL4 detection?

For optimal VGLL4 detection by immunohistochemistry:

• Antibody selection: Choose antibodies specifically validated for IHC applications. For example, product HPA038225 is recommended at dilutions of 1:50-1:200 for IHC applications .

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective for VGLL4 detection.

• Dilution optimization: Perform a titration experiment using different antibody dilutions (starting with manufacturer recommendations) on known positive control tissues.

• Controls:

  • Positive control: Normal lung tissue shows strong nuclear VGLL4 expression .

  • Negative control: Omit primary antibody or use tissue known to have low VGLL4 expression.

  • Specificity control: Use VGLL4 knockout or knockdown samples when available.

• Detection system: A polymer-based detection system generally provides better sensitivity and lower background compared to ABC methods.

• Counterstaining: Light hematoxylin counterstaining allows for better visualization of nuclear VGLL4 localization.

What are the critical considerations for Western blot analysis of VGLL4?

For successful Western blot detection of VGLL4:

• Protein extraction: Use RIPA buffer supplemented with protease inhibitors to prevent degradation during extraction.

• Sample preparation:

  • Load 25-30 μg of total protein per lane .

  • Include a reducing agent in sample buffer to break disulfide bonds.

• Gel selection: Use 10-12% SDS-PAGE gels, as VGLL4 has a calculated molecular weight of 31 kDa.

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 60-90 minutes.

  • Use PVDF membrane for better protein retention.

• Blocking:

  • 3-5% non-fat dry milk in TBST has been demonstrated to be effective .

  • Block for 1 hour at room temperature.

• Antibody dilution:

  • Primary: 1:500 - 1:2000 dilution is typically recommended .

  • Secondary: HRP-conjugated anti-rabbit IgG at 1:10,000 dilution .

• Detection: ECL-based detection systems with exposure times around 60 seconds are generally sufficient .

• Controls: Include positive control lysates from cells known to express VGLL4 (such as normal lung tissue extracts).

How can researchers effectively study VGLL4's role in cancer immune evasion?

To investigate VGLL4's role in cancer immune evasion, consider these methodological approaches:

• Syngeneic tumor models:

  • Use murine syngeneic models (like LLC lung cancer or MB49 bladder cancer cells) that comprise intact immune systems .

  • Compare tumor growth in Vgll4-knockdown versus control cells in both immune-competent and immune-compromised mice to distinguish between direct growth effects and immune-mediated effects .

• T-cell infiltration analysis:

  • Perform immunohistochemistry for leukocyte markers (CD45) and T-cell markers (CD3, CD8) to assess immune cell infiltration in VGLL4-deficient tumors .

  • Use flow cytometry to quantify the different immune cell populations within the tumor microenvironment.

• T-cell depletion experiments:

  • Deplete specific T-cell populations (CD4+, CD8+, or both) using blocking antibodies to determine which T-cell subset mediates tumor regression in VGLL4-deficient tumors .

  • The experimental data shows that "depletion of both CD8+ and CD4+ T cells yielded much larger tumor formation" in mice with Vgll4-knockdown tumors .

• PD-L1 expression analysis:

  • Measure PD-L1 expression by qRT-PCR, flow cytometry, and Western blot in VGLL4-manipulated cells .

  • Examine the response to IFNγ stimulation, as VGLL4 affects IFNγ-inducible PD-L1 expression .

• Promoter activity assays:

  • Use luciferase reporter assays with the PD-L1 promoter to directly assess VGLL4's effect on transcriptional regulation .

  • Research has shown that "PD-L1 promoter activity was significantly reduced in both INFγ-untreated and INFγ-treated VGLL4-knockdown A549 cells compared to control cells" .

What methodological approaches are best for studying VGLL4's interaction with the Hippo pathway?

To investigate VGLL4's interaction with the Hippo pathway components:

• Co-immunoprecipitation (Co-IP):

  • Use VGLL4 antibodies to pull down protein complexes and probe for TEAD family members.

  • Alternatively, use TEAD antibodies for immunoprecipitation and detect VGLL4 in the precipitates.

  • Include YAP in the analysis to examine competitive binding between YAP and VGLL4 to TEADs.

• Domain mapping:

  • Generate VGLL4 mutants (such as ΔTDUs and HF4A) to identify interaction domains.

  • Research has shown that "deletion of TDU domains or HF4A mutation in VGLL4 abolished the interaction between VGLL4 and TEAD4" .

• Transcriptional activity assays:

  • Use TEAD-responsive luciferase reporters to measure how VGLL4 affects YAP/TEAD-mediated transcription.

  • Compare wild-type VGLL4 with mutant versions that cannot bind TEADs.

• Liquid-liquid phase separation (LLPS) analysis:

  • Study how VGLL4 induces TEAD4 condensation by measuring droplet formation.

  • Research indicates that "VGLL4 triggers TEAD4 condensation by inducing its oligomerization" .

• ChIP-seq analysis:

  • Map TEAD4 genomic binding sites in the presence and absence of VGLL4.

  • Examine how VGLL4 affects the recruitment of transcriptional machinery to TEAD target genes.

How do researchers study the differential localization of VGLL4 in normal versus cancer tissues?

To effectively investigate VGLL4's subcellular localization patterns:

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions biochemically.

  • Analyze VGLL4 protein levels in each fraction by Western blot.

  • Include proper loading controls for each fraction (e.g., histone H3 for nuclear, GAPDH for cytoplasmic).

• Immunofluorescence microscopy:

  • Perform co-staining with nuclear markers (DAPI) and VGLL4 antibodies.

  • Use confocal microscopy for precise subcellular localization.

  • Research indicates VGLL4 "displayed a more diffused cytoplasmic staining in lung ADCs, but a predominant nuclear staining was seen in normal lungs" .

• Tissue microarray analysis:

  • Compare VGLL4 localization across multiple normal and cancer tissue samples.

  • Use quantitative image analysis to score nuclear versus cytoplasmic staining intensity.

  • In lung adenocarcinoma studies, "92.6% of patients (25 out of 27) exhibited high nuclear VGLL4 expression in their normal lungs, whereas only 22.1% of patients (17 out of 77) had high nuclear expression of VGLL4 in their lung ADCs" .

• Correlation with disease progression:

  • Analyze how VGLL4 localization changes with cancer stage and grade.

  • Correlate localization patterns with patient outcomes.

Why might researchers observe discrepancies between VGLL4 mRNA and protein levels?

Discrepancies between VGLL4 mRNA and protein levels can occur for several reasons:

• Post-transcriptional regulation:

  • miRNA-mediated regulation: Research has shown that "YAP inhibits IFNγ-inducible PD-L1 expression partially through miR-130a-mediated suppression of VGLL4" .

  • Alternative splicing: Up to 6 different isoforms have been reported for VGLL4 .

• Post-translational modifications:

  • Protein stability: VGLL4 may regulate protein stability of other factors (like IRF2BP2), but its own stability might also be regulated .

  • Proteasomal degradation: Like many transcription factors, VGLL4 might be subject to regulated degradation.

• Technical limitations:

  • Antibody specificity: Some antibodies may not detect all VGLL4 isoforms.

  • Protein extraction efficiency: Nuclear proteins can be more difficult to extract completely.

• Biological compartmentalization:

  • Subcellular localization changes: VGLL4 shows different localization patterns in normal versus cancer tissues .

  • Protein sequestration: VGLL4 may be sequestered in protein complexes that mask antibody epitopes.

To address these discrepancies, researchers should:

• Use multiple antibodies targeting different epitopes

• Validate findings with orthogonal methods (e.g., mass spectrometry)

• Consider analyzing both total and subcellular fraction levels

How can researchers address non-specific binding when using VGLL4 antibodies?

To minimize non-specific binding with VGLL4 antibodies:

• Antibody validation:

  • Use VGLL4 knockout or knockdown samples as negative controls.

  • Test antibodies on tissues known to express different levels of VGLL4.

  • Verify specificity using peptide competition assays, where the antibody is pre-incubated with the immunogen peptide.

• Blocking optimization:

  • Test different blocking agents: BSA, casein, normal serum from the secondary antibody host species.

  • Extend blocking time to 2 hours or overnight at 4°C for difficult samples.

  • Include 0.1-0.3% Triton X-100 in blocking buffer for intracellular antigens.

• Antibody dilution:

  • Optimize primary antibody concentration through titration experiments.

  • For Western blot, dilutions between 1:500-1:2000 are typically effective .

  • For immunohistochemistry, dilutions between 1:50-1:200 are recommended .

• Washing protocols:

  • Increase number and duration of washes.

  • Use TBS with 0.1% Tween-20 for more stringent washing.

  • Consider adding 0.05-0.1% SDS to wash buffer for very sticky antibodies.

• Secondary antibody considerations:

  • Pre-adsorb secondary antibodies against tissues from the species being studied.

  • Use highly cross-adsorbed secondary antibodies.

  • Consider using fluorescent secondaries for better signal-to-noise ratio in immunofluorescence.

How should researchers interpret contradictory findings regarding VGLL4's role in different cancer models?

When facing contradictory findings about VGLL4's role in cancer:

How can VGLL4's role in liquid-liquid phase separation (LLPS) be effectively investigated?

To study VGLL4's involvement in liquid-liquid phase separation:

• In vitro phase separation assays:

  • Purify recombinant TEAD4 and VGLL4 proteins.

  • Observe droplet formation using differential interference contrast (DIC) microscopy.

  • Research shows that "VGLL4 markedly enhanced formation of droplets of the TEAD4-DNA complex" .

• FRAP (Fluorescence Recovery After Photobleaching):

  • Tag TEAD4 and VGLL4 with fluorescent proteins.

  • Measure protein dynamics within condensates.

  • Compare recovery rates with and without VGLL4.

• DNA interaction studies:

  • Use EMSA (Electrophoretic Mobility Shift Assay) to assess how VGLL4 affects TEAD4-DNA interactions.

  • Research indicates that "a mixture of TEAD4 and VGLL4, but not a mixture of TEAD4Mut and VGLL4, held back the migration of the DNA segments" .

• Mutational analysis:

  • Test VGLL4 mutants for their ability to induce TEAD4 condensation.

  • The data shows that "VGLL4 triggers TEAD4 condensation by inducing its oligomerization, and that this process requires VGLL4's two TDU domains" .

• Chromatin organization:

  • Investigate how VGLL4-induced TEAD4 condensation affects chromatin accessibility.

  • Examine changes in histone modifications and transcriptional activity.

  • Research found that the "CTGF gene showed weaker CTCF, Pol II and H3K27ac signals in HEK293FT cells transfected with VGLL4 than in the negative control" .

What are the most promising therapeutic implications of VGLL4 research?

VGLL4 research presents several therapeutic opportunities:

• Cancer immunotherapy enhancement:

  • VGLL4 inhibition could potentially enhance anti-tumor immunity by reducing PD-L1 expression .

  • This approach might complement existing immune checkpoint inhibitors.

  • Research shows that "disruption of Vgll4 results in potent T cell-mediated tumor regression in murine syngeneic models" .

• Hippo pathway modulation:

  • Peptide mimetics of VGLL4's TDU domains could inhibit YAP-TEAD interactions.

  • This approach could suppress YAP-driven oncogenic programs.

  • VGLL4 "acts as a tumor suppressor in lung cancer through negatively regulating the YAP-TEAD complex formation" .

• Transcriptional condensate targeting:

  • Compounds that interfere with VGLL4-induced TEAD4 condensation could alter gene expression programs.

  • The research shows that 1,6-hexanediol treatment can reverse the effects of VGLL4 on TEAD4 condensation .

• Biomarker development:

  • VGLL4 expression and localization patterns could serve as prognostic biomarkers.

  • Human epidemiological data suggests that "lower expression of VGLL4 correlates with better patient outcome" .

  • Examining the nuclear-to-cytoplasmic ratio of VGLL4 might provide diagnostic value.

• Combinatorial approaches:

  • Understanding VGLL4's dual role in tumor growth and immune evasion could inform combination therapies.

  • Targeting both YAP and immune checkpoints might yield synergistic effects.

  • Research indicates the importance of considering both "tumor cell growth and immunogenicity" when targeting VGLL4 .