GPC4 Antibody

What is GPC4 and what are its key biological functions?

GPC4 (Glypican 4) is a cell surface proteoglycan that interacts with growth factors and signaling molecules to influence cellular processes including proliferation, migration, and differentiation . Also known as K-glypican, KPTS, dJ900E8.1, and glypican proteoglycan 4, GPC4 participates in multiple biological pathways . Functionally, GPC4 has been demonstrated to induce formation of active excitatory synapses by recruiting AMPA glutamate receptors to the postsynaptic cell surface . Additionally, it serves as a binding protein for bactericidal/permeability-increasing protein (BPI) in retinal pigment epithelial cells, mediating activation of ERK1/2 and Akt signaling pathways .

What applications are GPC4 antibodies typically used for in research?

GPC4 antibodies are employed across multiple research applications as outlined in the table below:

When selecting a GPC4 antibody, researchers should consider the validated applications and species reactivity provided by the manufacturer to ensure compatibility with their experimental design .

How can researchers validate the specificity of their GPC4 antibodies?

Antibody validation is crucial for ensuring experimental reliability. For GPC4 antibodies, researchers should implement the following validation strategies:

• Comparison with in situ hybridization data - As demonstrated in the Trimmer laboratory validation, comparing antibody staining patterns with in situ hybridization results from resources like the Allen Brain Atlas can confirm target specificity .

• Knockout/knockdown controls - Using GPC4 knockout tissues or cells with GPC4 siRNA knockdown as negative controls can verify antibody specificity.

• Peptide competition assays - Pre-incubating the antibody with recombinant GPC4 protein (particularly using the immunogen sequence corresponding to amino acids 357-556 of human GPC4) should abolish specific staining .

• Cross-reactivity testing - Testing against related glypicans (GPC1-6) ensures the antibody specifically recognizes GPC4 and not its family members.

How do different epitope targets affect GPC4 antibody performance in various applications?

The epitope target significantly impacts antibody functionality across different applications. The amino acid sequence targeted by an antibody determines its ability to recognize native, denatured, or post-translationally modified GPC4.

Antibodies targeting the extracellular domain (such as those recognizing amino acids 88-101) have demonstrated efficacy in both Western blot and frozen section immunohistochemistry applications . Conversely, antibodies targeting the sequence corresponding to amino acids 357-556 of human GPC4 show reactivity in both human and mouse samples for Western blot and ELISA applications .

For functional studies investigating GPC4's role in signaling pathways, antibodies targeting the core protein rather than the glycosaminoglycan chains are preferred as they can effectively block protein-protein interactions. When studying GPC4's interactions with AMPA receptors or neuronal pentraxins, researchers should select antibodies that don't interfere with the binding domains involved in these interactions .

What methodological approaches can distinguish between membrane-bound and secreted forms of GPC4?

Distinguishing between membrane-bound and secreted GPC4 requires specific methodological approaches:

• Cell fractionation combined with Western blotting - Separate membrane fractions from culture media/supernatant and compare GPC4 levels in each fraction.

• Enzymatic treatments - Use phosphatidylinositol-specific phospholipase C to cleave GPI anchors, releasing membrane-bound GPC4. The effectiveness of this treatment in modulating GPC4-dependent signaling has been demonstrated in bovine retinal pigment epithelial cells .

• Heparitinase treatment - This enzyme degrades heparan sulfate chains on GPC4, which can affect its signaling capabilities. Research shows that heparitinase suppresses BPI-induced ERK and Akt phosphorylation in bovine RPE and inhibits BPI actions on VEGF and PDGF-B mRNA expression induced by H₂O₂ .

• Immunoprecipitation assays - Using antibodies specific to post-translational modifications unique to either membrane-bound or secreted forms.

Studies in neuronal systems have shown that soluble GPC4 protein can mimic the astrocyte-secreted form, inducing synapse formation through regulation of NP1 (Neuronal Pentraxin 1) release and surface accumulation .

How can researchers investigate GPC4's interactions with its signaling partners?

To investigate GPC4's interactions with signaling partners, researchers can employ the following methodologies:

• Co-immunoprecipitation - Using GPC4 antibodies to pull down protein complexes, followed by immunoblotting for suspected binding partners. This approach was instrumental in identifying GPC4 as a binding protein for BPI in retinal cells .

• Proximity ligation assays - Enabling visualization of protein-protein interactions in situ with spatial resolution below 40 nm.

• Surface plasmon resonance - Quantifying binding kinetics between purified GPC4 and potential partners.

• Functional pathway analysis - Monitoring downstream signaling events following GPC4 manipulation. Research has shown that GPC4 activates ERK1/2 and Akt signaling pathways in bovine RPE cells, and these effects can be suppressed by anti-GPC4 antibody treatment . Additionally, GPC4 has been demonstrated to regulate neuronal signaling pathways that mediate AMPAR dendritic clustering and increase the extracellular levels of NP1 (2.07-fold ± 0.10-fold) and NP2 (1.41-fold ± 0.10-fold) .

What are the optimal fixation and sample preparation protocols for GPC4 immunohistochemistry?

Successful GPC4 immunohistochemistry depends on proper sample preparation:

For brain tissue samples, the following protocol has been validated:

• Fixation: Formaldehyde/paraformaldehyde fixation preserves tissue architecture while maintaining GPC4 antigenicity .

• Sample preparation: Free-floating sections work well for brain tissue analysis .

• Permeabilization: PBS containing 1:2000 avidin and 0.5% Triton X-100 for 45 minutes at room temperature facilitates antibody penetration .

• Blocking: 0.5% serum for 45 minutes at room temperature effectively reduces non-specific binding .

• Primary antibody incubation: GPC4 antibody at 1 μg/mL concentration, overnight at 4°C .

• Secondary antibody: Biotinylated goat anti-rabbit IgG (H+L) at 1 μg/mL for 60 minutes at room temperature .

This protocol has been validated by comparing staining patterns to expression levels from in situ hybridization performed by the Allen Brain Atlas on sagittal sections of P56 mouse brain .

How should researchers design controls when using GPC4 antibodies in functional studies?

Proper experimental controls are essential for GPC4 antibody studies:

• Blocking peptide controls - Pre-incubate the GPC4 antibody with excess immunizing peptide to confirm signal specificity.

• Isotype controls - Use matched isotype IgG at the same concentration as the GPC4 antibody to assess non-specific binding.

• Genetic controls - Include GPC4 knockout or knockdown samples alongside wild-type samples.

• Enzymatic controls - As demonstrated in studies with retinal pigment epithelial cells, treatments with heparitinase or phosphatidylinositol-specific phospholipase C can serve as functional controls by disrupting GPC4-dependent signaling .

• Biological activity validation - Compare antibody effects to known GPC4 biological activities, such as its ability to induce active synapse formation in retinal ganglion cells or regulate NP1 secretion .

What quantification methods are most appropriate for measuring GPC4 expression changes?

Appropriate quantification methods depend on the experimental approach:

• Western blot densitometry - For relative protein level comparison across samples, normalize GPC4 band intensity to housekeeping proteins.

• qRT-PCR - For transcript level analysis, given that GPC4 overexpression has been shown to affect mRNA levels of various cell cycle regulators, including cyclin D isoforms .

• Immunofluorescence quantification - For tissue or cellular localization studies:

  • Mean fluorescence intensity measurements in defined regions

  • Co-localization coefficients when studying GPC4 with binding partners

  • Surface-to-total protein ratio when investigating membrane versus intracellular distribution

• Flow cytometry - For cell surface expression analysis, permitting high-throughput quantification on a per-cell basis.

• Time course experiments - As demonstrated in studies of NP1 surface accumulation, where measurements were performed at multiple time points (4h, 12h) to track the temporal dynamics of GPC4-mediated effects .

How should researchers interpret apparent contradictions in GPC4 antibody results across different experimental systems?

When facing contradictory GPC4 antibody results, researchers should consider:

• Isoform specificity - Different antibodies may detect distinct GPC4 isoforms or post-translational modifications.

• Context-dependent expression - GPC4 function and expression can vary significantly between cell types. For example, GPC4's effects on cell signaling differ between neurons and retinal pigment epithelial cells .

• Technical variables - Differences in sample preparation, antibody concentration, incubation time, and detection methods can all influence results. The Trimmer lab protocol for immunohistochemistry provides a standardized approach that can minimize such variability .

• Biological complexity - GPC4 may have dual or even opposing functions depending on the cellular context and binding partners present. For instance, GPC4 can both impair cyclin D-CDK4/6 kinases and promote other signaling pathways such as Akt/MAPK .

• Species differences - Consider potential variations in GPC4 structure and function across species when comparing results from human, mouse, rat, or bovine systems.

What factors should be considered when analyzing GPC4's role in cell signaling pathways?

When analyzing GPC4's role in signaling pathways, researchers should consider:

• Temporal dynamics - GPC4-induced effects may have different time courses. For example, its effect on GluA1 synaptic expression is relatively slow, while effects on surface NP1 can be detected as early as 4 hours after treatment (1.72-fold ± 0.17-fold increase) .

• Upstream and downstream effectors - GPC4 interacts with multiple signaling molecules. In retinal pigment epithelial cells, it serves as a binding protein for BPI, mediating activation of ERK1/2 and Akt . In neurons, it regulates AMPAR dendritic clustering through effects on neuronal pentraxins .

• Cell-specific contexts - GPC4 can have different effects depending on the cellular environment. Its impact on cell cycle regulation in endothelial cells differs from its effects on synapse formation in neurons .

• Possible compensation by other glypicans - The glypican family consists of six members (GPC1-6) with potential functional overlap.

• Interaction with extracellular matrix components - As a cell surface proteoglycan, GPC4's signaling functions may be influenced by its interactions with matrix proteins.

How can GPC4 antibodies be utilized to study its role in pathological conditions?

GPC4 antibodies enable several approaches to studying its role in disease:

• Expression pattern analysis - Compare GPC4 levels in normal versus diseased tissues using immunohistochemistry and Western blot. Dysregulation of GPC4 expression has been implicated in cancer, cardiovascular disorders, and developmental abnormalities .

• Signaling pathway investigation - Using GPC4 antibodies to block its function can reveal its contribution to pathological signaling. For example, anti-GPC4 antibody has been shown to suppress BPI-induced ERK and Akt phosphorylation in bovine RPE .

• Co-localization studies - Examine GPC4's association with disease-related proteins in tissue samples.

• Biomarker potential assessment - Evaluate whether GPC4 levels correlate with disease progression or treatment response using quantitative immunoassays.

• Therapeutic target validation - Determine whether blocking GPC4 with antibodies affects disease-related phenotypes in cellular or animal models.

What methodological considerations are important when studying GPC4 in neurological disorders?

When investigating GPC4 in neurological disorders, researchers should consider:

• Brain region specificity - GPC4 expression varies across brain regions, necessitating precise anatomical sampling. Validation protocols comparing antibody staining to in situ hybridization data from resources like the Allen Brain Atlas can help ensure accurate localization .

• Cell-type specificity - Distinguish between neuronal, astrocytic, and other cell sources of GPC4. Research has shown that astrocyte-secreted GPC4 induces formation of active excitatory synapses by recruiting AMPA glutamate receptors .

• Functional assays - Beyond expression studies, assess GPC4's functional impact using electrophysiology to measure synaptic transmission, as GPC4 regulates AMPAR trafficking to synapses .

• Developmental timing - Consider GPC4's changing roles throughout neurodevelopment when designing studies.

• Model system selection - Choose models (primary cultures, organoids, animal models) that best recapitulate the human pathology being studied.

• Interaction with disease mechanisms - Investigate how GPC4 interacts with known disease mechanisms, such as its potential impact on neuronal signaling pathways that might be dysregulated in neurological disorders .

What are the best practices for using GPC4 antibodies in cancer research?

In cancer research, GPC4 antibody applications should follow these best practices:

• Tissue microarray analysis - Screen GPC4 expression across multiple tumor samples and correlate with clinical outcomes.

• Cell line validation - Confirm antibody specificity in relevant cancer cell lines before proceeding to complex tumor samples.

• Functional blocking studies - Use antibodies to neutralize GPC4 function and observe effects on cancer cell proliferation, migration, and invasion, particularly given GPC4's known roles in regulating cell proliferation and signaling pathways .

• Signaling pathway analysis - Investigate GPC4's impact on cancer-relevant signaling, such as its effects on cell cycle regulators. Studies have shown that GPC1 (another glypican family member) can impair cyclin D-CDK4/6 kinases, suggesting potential similar mechanisms for GPC4 .

• Co-expression studies - Examine GPC4's relationship with known oncogenes or tumor suppressors using multiplex immunofluorescence.

• Therapeutic response prediction - Determine whether GPC4 expression levels correlate with response to specific cancer therapies.

• Patient stratification - Assess whether GPC4 expression or localization pattern can serve as a biomarker for patient stratification in clinical trials.