DLG5 Antibody

What is DLG5 and why is it important in cellular research?

DLG5 (Discs Large Homolog 5) is a member of the membrane-associated guanylate kinase (MAGUK) family that serves as a critical molecular scaffold. It primarily maintains the structural integrity of epithelial cell plasma membranes and facilitates multiprotein complex assembly at cell junctions . Research has identified DLG5 as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, demonstrating direct connections between cell polarity and intracellular signaling pathways .

DLG5 plays significant roles in:

• Transmitting extracellular signals to the cytoskeleton

• Maintaining apical-basal polarity in epithelial cells

• Regulating branching morphogenesis during development

• Supporting proper ciliation in multiple tissues

• Influencing progenitor cell differentiation

This multifunctionality makes DLG5 a critical target for developmental biology, cancer research, and studies of congenital disorders.

How do I select the appropriate DLG5 antibody for my research application?

Selection requires careful consideration of multiple factors:

• Research application: Determine which applications (WB, IP, IF, ELISA) are central to your research. Available antibodies have different validation profiles:

  • For Western blotting: Multiple antibodies show reactivity (15687-1-AP, sc-374493, sc-374594)

  • For immunoprecipitation: Consider monoclonal antibodies like E-11 (sc-374493) or A-11 (sc-374594)

  • For immunofluorescence: Both polyclonal and monoclonal options are available

• Species reactivity: Confirm the antibody recognizes DLG5 in your model organism. Currently available antibodies recognize:

  • Human DLG5 (most antibodies)

  • Mouse DLG5 (sc-374493, sc-374594, 15687-1-AP)

  • Rat DLG5 (sc-374493, sc-374594)

• Antibody type: Consider whether monoclonal specificity or polyclonal broader epitope recognition better suits your needs:

  • Monoclonal options: E-11 (sc-374493) and A-11 (sc-374594)

  • Polyclonal options: 15687-1-AP

• Isoform specificity: Some antibodies target specific DLG5 isoforms, such as sc-374594 which detects isoforms 1, 2, and 4 .

• Validation data: Review available validation through knockdown experiments, independent antibody comparisons, or tagged protein localization studies .

What are the optimal protocols for detecting DLG5 in Western blot applications?

For successful Western blot detection of DLG5:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for whole cell extracts

  • For membrane-enriched fractions (recommended since DLG5 is membrane-associated), consider membrane protein extraction kits

  • Include phosphatase inhibitors if studying DLG5 phosphorylation states

• Protein loading and separation:

  • Load 20-40 μg of total protein (adjust based on expression level)

  • Use 6-8% SDS-PAGE gels due to DLG5's large size (209 kDa calculated molecular weight)

  • Consider gradient gels (4-15%) for better resolution of both full-length and potential cleaved products

• Transfer considerations:

  • Extend transfer time (overnight at lower voltage) for efficient transfer of large proteins

  • Use PVDF membranes rather than nitrocellulose for higher binding capacity

• Antibody dilutions and detection:

  • Primary antibody: Use 1:500-1:1000 dilution for optimal results

  • Secondary antibody: HRP-conjugated anti-mouse or anti-rabbit IgG at 1:5000-1:10000

  • Consider enhanced chemiluminescence detection systems for optimal sensitivity

• Molecular weight verification:

  • Full-length DLG5 appears at approximately 240 kDa

  • A smaller isoform/fragment is often observed at approximately 75 kDa

  • Verify specificity based on calculated molecular weight (209 kDa)

• Positive controls:

  • PC-3 cells, HeLa cells, and mouse kidney tissue have been verified as positive controls

How can I optimize immunofluorescence protocols for DLG5 detection in different tissue types?

For effective DLG5 immunofluorescence staining:

• Fixation optimization:

  • For cell lines: 4% paraformaldehyde (10-15 minutes) preserves membrane structures

  • For tissues: Consider 4% PFA perfusion followed by post-fixation (4-24 hours depending on tissue)

  • Alternative: Methanol fixation (-20°C, 10 minutes) can improve access to certain epitopes

• Antigen retrieval methods:

  • For FFPE tissues: Heat-induced epitope retrieval with TE buffer (pH 9.0) is recommended

  • Alternative: Citrate buffer (pH 6.0) can be used as an alternative

  • For frozen sections: Milder retrieval methods may be sufficient

• Antibody dilution and incubation:

  • Primary antibody: 1:50-1:500 dilution range

  • Incubation: Overnight at 4°C for optimal signal-to-noise ratio

  • Include appropriate blocking steps (5% normal serum + 0.3% Triton X-100)

• Co-staining strategies:

  • Membrane markers (E-cadherin) to visualize cell-cell junctions

  • Apical markers (aPKC) to examine apical-basal polarity

  • Ciliary markers when studying DLG5's role in ciliogenesis

• Tissue-specific considerations:

  • Lung tissue: DLG5 localizes to epithelial cell junctions and is important in branching morphogenesis

  • Brain tissue: Focus on ventricular zones where DLG5 influences neuroprogenitor development

  • Kidney tissue: Pay attention to tubular structures where DLG5 affects cilia formation

• Controls:

  • Positive tissue controls: Prostate and placenta show high DLG5 expression

  • Negative controls: Include secondary-only controls and tissues from DLG5 knockout models when available

How can I study DLG5's interactions with Hippo signaling components in cellular models?

To investigate DLG5-Hippo pathway interactions:

• Co-immunoprecipitation approach:

  • Use DLG5 antibodies (sc-374493 AC for immunoprecipitation) to pull down protein complexes

  • Probe for known interactors: MST1/2, SAV1, and MARK1/2/3

  • Perform reciprocal co-IPs with antibodies against Hippo pathway components

  • Use embryonic neural progenitor cells as a relevant model system

• Proximity ligation assay:

  • Visualize endogenous protein-protein interactions in situ

  • Combine DLG5 antibody with antibodies against Hippo pathway components

  • Quantify interaction signals in different cellular compartments

• Domain mapping experiments:

  • Create constructs expressing specific DLG5 domains: CARD, coiled-coil, PDZ, SH3, and GUK domains

  • Test binding affinities to MST1/2 using pulldown assays

  • Identify critical residues mediating interactions through mutagenesis

• Functional assays:

  • Monitor MST1/2 kinase activity in the presence/absence of DLG5 using phospho-specific antibodies

  • Assess YAP/TAZ nuclear localization as a downstream readout of Hippo pathway activity

  • Compare cells with DLG5 knockdown or overexpression to observe pathway modulation

• Experimental controls:

  • Include DLG5-knockout cells as negative controls

  • Use IgG antibodies as non-specific binding controls

  • Generate rescue experiments with wild-type and mutant DLG5 constructs

Research has confirmed that DLG5 negatively regulates Hippo signaling by inhibiting MST1/2 association with LATS1/2 and recruiting MARK3 to MST1/2, resulting in hyperphosphorylation and inhibition of MST1/2 kinase activity .

What approaches can help distinguish DLG5's role in ciliogenesis versus cell polarity maintenance?

To differentiate these interconnected functions:

• Temporal analysis of protein localization:

  • Perform time-course immunofluorescence during ciliogenesis

  • Track DLG5 localization relative to polarity markers (aPKC, Par complex) and ciliary markers

  • Use live-cell imaging with fluorescent-tagged DLG5 to monitor dynamic localization

• Domain-specific mutant analysis:

  • Generate constructs with mutations in specific DLG5 domains

  • Test rescue capabilities in DLG5-depleted cells for:

    • Apical-basal polarity markers restoration

    • Cilia formation and function

    • Distinguish mutations that affect one function but not the other

• Tissue-specific analyses:

  • Compare DLG5 knockout effects across tissues:

    • Kidney: Focus on cilia in tubular structures

    • Brain: Examine ependymal cell layer and ventricular development

    • Lung: Assess branching morphogenesis and epithelial polarity

• Molecular pathway dissection:

  • Use siRNA to independently knockdown polarity proteins versus cilia-specific proteins

  • Determine if DLG5's effects on cilia require intact polarity machinery

  • Examine if forced localization of DLG5 to specific cellular compartments rescues distinct functions

• Disease-associated variant analysis:

  • Test patient-derived DLG5 variants in rescue experiments

  • Compare their ability to restore:

    • Proper ciliation in kidney and brain tissues

    • Tissue morphology

    • Research has shown patient variants were largely ineffective in restoring both ciliation and tissue morphology

This approach can help determine whether DLG5's role in ciliogenesis is a direct mechanism or an indirect consequence of its function in establishing cell polarity.

How can I address the challenge of detecting multiple DLG5 isoforms with consistent specificity?

DLG5 is known to express multiple isoforms with observed molecular weights from 75 kDa to 240 kDa , creating detection challenges:

• Isoform-specific detection strategies:

  • Select antibodies validated for specific isoforms (e.g., sc-374594 for isoforms 1, 2, and 4)

  • Design PCR primers to distinguish transcript variants before protein analysis

  • Consider using tissue-specific positive controls where particular isoform expression is well-documented

• Optimization for high molecular weight proteins:

  • For full-length DLG5 (~240 kDa):

    • Use low percentage (6%) or gradient gels

    • Extended transfer times (overnight at low voltage or semi-dry high amp protocols)

    • Methanol-free transfer buffers can improve transfer of large proteins

• Validation approaches:

  • siRNA knockdown validation: Target regions common to all isoforms and assess which bands decrease

  • Blocking peptides: Use peptides corresponding to the antibody epitope to confirm specificity

  • Compare results from antibodies targeting different DLG5 epitopes

• Sample preparation considerations:

  • Test different lysis buffers to ensure complete extraction of membrane-associated proteins

  • Include protease inhibitors to prevent degradation that might be misinterpreted as isoforms

  • Fresh preparation versus frozen samples may yield different isoform patterns

• Data analysis guidelines:

  • Always report molecular weights of observed bands

  • Specify which isoform is being studied in your research

  • Consider the tissue context when interpreting isoform patterns (e.g., placenta and prostate show distinct expression profiles)

What strategies help resolve conflicting results when studying DLG5 in different cell types or developmental stages?

When facing contradictory findings in DLG5 research:

• Context-dependent function analysis:

  • DLG5 shows tissue-specific roles in branching morphogenesis , ciliogenesis , and Hippo signaling

  • Map protein interaction networks in each cell type to identify tissue-specific partners

  • Compare DLG5 subcellular localization across cell types using the same validated antibody

• Developmental timing considerations:

  • Track DLG5 expression and localization at multiple developmental stages

  • When comparing studies, note exact developmental timepoints:

    • For example, DLG5 knockout shows minimal differences at E12-E12.5 but prominent defects by E13.5 in lung development

  • Use inducible knockout systems to distinguish acute versus developmental phenotypes

• Cross-validation with multiple approaches:

  • Combine genetic models (knockout, knockdown) with biochemical approaches

  • Verify findings using both in vivo and ex vivo systems

  • Test phenotypes in multiple species (mouse models, Xenopus embryos, etc.)

• Data reconciliation framework:

  • Systematically document experimental variables when comparing studies:

    • Antibody used (clone, lot, dilution)

    • Tissue preparation method

    • Animal strain or cell line specifics

  • Create side-by-side comparisons under identical conditions

• Rescue experiment design:

  • Test if wild-type DLG5 rescues knockout phenotypes in your specific context

  • Compare rescue efficiency of different DLG5 variants or truncated constructs

  • Patient variants have shown different rescue capabilities across tissues, suggesting context-dependent functions

How can DLG5 antibodies be applied to study ciliopathies and congenital developmental disorders?

Recent research has implicated DLG5 in ciliopathies and congenital anomalies , offering new applications:

• Clinical sample analysis protocol:

  • Examine DLG5 expression and localization in patient biopsies

  • Compare control versus patient tissues using validated antibodies (15687-1-AP for IHC, 1:50-1:500 dilution)

  • Focus on tissues with known ciliopathy manifestations:

    • Kidney: Examine cystic regions and tubular structures

    • Brain: Focus on ependymal lining of ventricles

    • Limbs: Analyze growth plate organization

• Variant characterization methodology:

  • Immunostaining to assess protein localization of patient-derived DLG5 variants

  • Functional analyses using cellular models and rescue experiments

  • DLG5 variants have been associated with cystic kidneys, nephrotic syndrome, hydrocephalus, limb abnormalities, and congenital heart disease

• Animal model applications:

  • Use antibodies to track DLG5 expression in animal models of ciliopathies

  • Compare protein localization across developmental stages

  • Xenopus embryos depleted of dlg5 recapitulate many patient phenotypes and show loss of cilia in multiple tissues

• Diagnostic potential assessment:

  • Evaluate DLG5 antibodies for potential diagnostic applications in ciliopathies

  • Develop immunostaining protocols optimized for clinical samples

  • Consider multi-marker panels combining DLG5 with established ciliopathy markers

• Therapeutic target validation:

  • Use antibodies to monitor DLG5 modulation in response to candidate therapeutics

  • Track restoration of proper DLG5 localization and downstream effectors

  • Assess correlation between DLG5 regulation and phenotypic rescue

What methodological approaches can be used to investigate DLG5's role in cancer progression and metastasis?

DLG5 expression is decreased in multiple cancers, including bladder, prostate, breast, and hepatocellular carcinoma , suggesting important research applications:

• Expression analysis in cancer tissues:

  • Optimize IHC protocols for cancer tissue microarrays

  • Recommended antibody dilutions: 1:50-1:500 for IHC applications

  • Correlate DLG5 expression patterns with clinical parameters and survival data

• Mechanistic investigation workflow:

  • Establish stable DLG5 knockdown and overexpression cancer cell lines

  • Assess effects on:

    • Cell proliferation and apoptosis markers

    • Cell migration and invasion assays

    • Epithelial-mesenchymal transition markers

  • DLG5 loss has been linked to activation of cell invasion and metastasis in prostate and bladder cancers

• Pathway interaction analysis:

  • Investigate DLG5's relationship with known cancer pathways:

    • Hippo signaling (DLG5 negatively regulates MST1/2)

    • Hedgehog signaling (DLG5 has been shown to regulate this pathway)

    • Cell polarity regulation (disrupted in many cancers)

  • Use co-immunoprecipitation with antibodies like sc-374493 AC to pull down cancer-relevant interacting partners

• In vivo metastasis model design:

  • Apply DLG5 antibodies for IHC analysis of metastatic tissues

  • Compare primary versus metastatic lesions for DLG5 expression patterns

  • Correlate changes with epithelial-mesenchymal transition markers

• Therapeutic response monitoring:

  • Track DLG5 expression and localization in response to cancer therapies

  • Investigate whether restoring DLG5 expression sensitizes cancer cells to treatments

  • Develop combination approaches targeting DLG5-regulated pathways