SH3PXD2A Antibody

What is SH3PXD2A and what are its key structural features?

SH3PXD2A (also known as TKS5, FISH, or SH3MD1) is a scaffold protein containing one phox homology (PX) domain in the N-terminal region followed by five SRC homology 3 (SH3) domains. The protein has a calculated molecular weight of 125 kDa, though it typically appears at 140-150 kDa in western blots due to post-translational modifications . The PX domain binds to membrane phospholipids including phosphatidylinositol-3-phosphate (PtdIns3P) and PtdIns(3,4)P2, while the SH3 domains mediate protein-protein interactions with partners involved in cytoskeletal remodeling and cell migration .

SH3PXD2A contains two SRC kinase phosphorylation sites at tyrosine 558 and 620, which are crucial for its activation and membrane localization . The protein exists in multiple isoforms, with p140 and p130 forms potentially generated by alternative splicing .

SH3PXD2A antibodies have been successfully tested in the following cell lines:

• HeLa cells (human cervical cancer cell line)

• HepG2 cells (human liver cancer cell line)

• A549 cells (human lung adenocarcinoma cells)

• H1299, H292, and H23 cells (human lung cancer cell lines)

• SKOV3 cells (human ovarian cancer cell line)

• ES2 cells (human ovarian cancer cell line)

These validations provide researchers with confidence in using these cell lines for SH3PXD2A studies .

How does SH3PXD2A contribute to invadopodia formation and cancer cell migration?

SH3PXD2A plays a critical role in invadopodia formation and cancer cell migration through multiple mechanisms:

• Membrane phospholipid binding: Upon cytokine stimulation, SRC kinase phosphorylates SH3PXD2A, enabling its PX domain to bind PtdIns3P and PtdIns(3,4)P2 in the cell membrane, anchoring the protein complex at invadopodia sites .

• Cytoskeletal reorganization: The SH3 domains of SH3PXD2A interact with proteins like WASL/N-WASP, GRB2, and NCK2 to remodel actin at invadopodia regions .

• Matrix metalloproteinase trafficking: SH3PXD2A transports MMP2-, MMP9-, and MMP14-containing vesicles to invadopodia and facilitates their release into the extracellular matrix, promoting degradation of extracellular matrix components and enabling cell invasion .

Research has demonstrated that knockdown of SH3PXD2A significantly reduces invadopodia formation, cell invasion, and metastasis in multiple cancer types . Site-directed mutagenesis experiments have shown that mutation of phosphorylation sites in SH3PXD2A (SH3PXD2A-[6A]) decreases its binding to PtdIns3P and reduces MMP14 activity, confirming the importance of these phosphorylation events for SH3PXD2A function in invadopodia formation .

What is the relationship between SH3PXD2A and SH3PXD2A-AS1 in cancer progression?

SH3PXD2A-AS1 is a long noncoding RNA (lncRNA) that has been found to be upregulated in non-small cell lung carcinoma (NSCLC) compared to normal lung tissues . While SH3PXD2A is a protein involved in invadopodia formation and cell migration, SH3PXD2A-AS1 appears to function through different mechanisms:

• Interaction with DHX9: SH3PXD2A-AS1 has been shown to interact with the DHX9 protein to enhance FOXM1 expression in NSCLC cells .

• Effect on cell proliferation: Overexpression of SH3PXD2A-AS1 promotes cell growth and proliferation of lung cancer cells, while knockdown significantly inhibits growth and proliferation .

• Cell cycle regulation: SH3PXD2A-AS1 influences cell cycle progression, with upregulation increasing the percentage of cells in S/G2 phases and downregulation decreasing this percentage .

Western Blot (WB)

• Sample preparation: Total protein extracts from cells or tissues

• Recommended dilution: 1:500-1:2,000

• Expected molecular weight: 140-150 kDa (although calculated MW is 125 kDa)

• Positive controls: HeLa cells, HepG2 cells

Immunohistochemistry (IHC)

• Sample preparation: Formalin-fixed, paraffin-embedded tissues

• Antigen retrieval: Use TE buffer pH 9.0 or citrate buffer pH 6.0

• Recommended dilution: 1:20-1:200

• Positive controls: Human breast cancer tissue

• Staining pattern: Cytoplasmic

Immunofluorescence (IF/ICC)

• Sample preparation: For cell fixation, use 4% paraformaldehyde followed by immersion in ice-cold acetone for 20 min at -20°C

• Blocking: Use blocking buffer for 1h at room temperature

• Recommended dilution: 1:10-1:100

• Positive controls: HepG2 cells

• Detection: Use appropriate fluorescent secondary antibodies (e.g., Alexa Fluor 488, Alexa Fluor 549)

• Imaging: Confocal laser scanning microscopy

How does SH3PXD2A phosphorylation affect its function in cancer cells?

SH3PXD2A phosphorylation is critical for its function in cancer cells:

• Activation mechanism: SH3PXD2A contains two SRC kinase phosphorylation sites at tyrosine 558 and 620. Upon cytokine stimulation, SRC kinase is activated and phosphorylates these sites .

• Membrane localization: Phosphorylation enhances SH3PXD2A binding to membrane phospholipids (PtdIns3P and PtdIns(3,4)P2), which is essential for docking the SH3PXD2A complex at invadopodia regions .

• MMP recruitment: Phosphorylated SH3PXD2A facilitates the transport and accumulation of MMP14 (MT1-MMP) in invadopodia regions, enhancing extracellular matrix degradation capacity .

Experimental evidence from site-directed mutagenesis demonstrates that mutation of phosphorylation sites (SH3PXD2A-[6A]) significantly decreases binding to PtdIns3P and reduces active MMP14 recruitment compared to wild-type SH3PXD2A . Zymography assays confirm that SH3PXD2A-[6A] mutant complexes show lower MMP14 activity than wild-type SH3PXD2A complexes .

These findings highlight the critical role of SH3PXD2A phosphorylation in cancer cell invasion and metastasis, suggesting potential therapeutic strategies targeting this regulatory mechanism.

What are the best practices for sample preparation when using SH3PXD2A antibodies?

For optimal detection of SH3PXD2A using antibodies, the following sample preparation protocols are recommended:

• Cell lysate preparation for Western blot:

  • Harvest cells at 70-80% confluence

  • Lyse cells in buffer containing protease inhibitors and phosphatase inhibitors (crucial for preserving phosphorylated forms)

  • Clear lysates by centrifugation (14,000 × g for 10 minutes at 4°C)

  • Determine protein concentration using a compatible assay (e.g., BCA)

  • Denature samples in Laemmli buffer (containing SDS and β-mercaptoethanol)

  • Heat at 95°C for 5 minutes

• Tissue preparation for IHC:

  • Fix tissues in 4% paraformaldehyde or 10% neutral buffered formalin

  • Embed in paraffin and section at 4-6 μm thickness

  • Perform antigen retrieval using TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

  • Block with appropriate blocking buffer before antibody incubation

• Cell preparation for IF/ICC:

  • Grow cells on coverslips to appropriate confluence

  • Fix with 4% paraformaldehyde

  • Immerse in ice-cold acetone for 20 minutes at -20°C

  • Block with appropriate blocking buffer for 1 hour at room temperature before antibody incubation

• Storage of samples and antibodies:

  • Store antibodies at -20°C for long-term storage

  • For frequent use, aliquot and store at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles

  • Store cell/tissue lysates at -80°C

How can researchers troubleshoot common issues with SH3PXD2A detection?

When encountering problems detecting SH3PXD2A, consider the following troubleshooting approaches:

• Weak or no signal in Western blot:

  • Verify protein transfer efficiency using a reversible stain

  • Increase antibody concentration or incubation time

  • Ensure fresh samples with intact protein (add protease inhibitors)

  • Increase loading amount (SH3PXD2A observed between 140-150 kDa despite calculated MW of 125 kDa)

  • Try alternative lysis buffers that better preserve membrane-associated proteins

• High background in IHC/IF:

  • Optimize blocking conditions (time, temperature, blocking agent)

  • Dilute primary antibody further (test range from 1:10-1:200 for IF, 1:20-1:200 for IHC)

  • Reduce secondary antibody concentration

  • Include additional washing steps

  • Use verified positive controls (HepG2 cells for IF, human breast cancer tissue for IHC)

• Inconsistent band size:

  • Note that SH3PXD2A typically appears at 140-150 kDa despite calculated MW of 125 kDa

  • Different isoforms (p140 and p130) may be detected

  • Phosphorylation status affects migration pattern

  • Consider using phosphatase treatment to determine contribution of phosphorylation to observed MW

• Cross-reactivity concerns:

  • Validate specificity using knockout/knockdown controls

  • Use multiple antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

What experimental designs are recommended for studying SH3PXD2A in cancer models?

For comprehensive investigation of SH3PXD2A in cancer models, consider these experimental designs:

• Expression analysis:

  • Compare SH3PXD2A protein levels between tumor and normal matched tissues using Western blot and IHC

  • Analyze SH3PXD2A expression across cancer cell lines representing different stages or grades

  • Correlate expression with clinical parameters and patient outcomes

• Functional studies:

  • Generate stable knockdown and overexpression models:

    • Use lentivirus-mediated shRNA for knockdown

    • Use full-length recombinant plasmids for overexpression

  • Validate knockdown/overexpression efficiency by qRT-PCR and Western blot

  • Assess functional outcomes using:

    • Proliferation assays (CCK-8, colony formation)

    • Cell cycle analysis by flow cytometry

    • Migration and invasion assays

    • Invadopodia formation assays

    • Matrix degradation assays

    • In vivo tumor growth and metastasis models

• Mechanism investigation:

  • Protein-protein interaction studies:

    • Co-immunoprecipitation to identify binding partners

    • RNA pulldown assays if investigating lncRNA interactions

    • RIP (RNA immunoprecipitation) assays for RNA-protein interactions

  • Phosphorylation analysis:

    • Site-directed mutagenesis of phosphorylation sites

    • Phospholipid binding assays

    • Zymography for MMP activity assessment

  • Localization studies:

    • Confocal microscopy to determine co-localization with other proteins or membrane structures

• Clinical correlations:

  • Analyze SH3PXD2A expression in patient-derived xenografts

  • Correlate expression with metastatic potential

  • Assess relationship with treatment response

When designing these experiments, include appropriate controls:

• Positive controls (HeLa or HepG2 cells for Western blot)

• Negative controls (cell lines with known low expression)

• Internal loading controls for Western blots

• Isotype controls for immunoprecipitation studies

What are the emerging roles of SH3PXD2A in cancer biology beyond invadopodia formation?

Recent research has uncovered additional roles for SH3PXD2A in cancer biology beyond its established function in invadopodia formation:

• Autophagy regulation: Evidence suggests a functional relationship between SH3PXD2A and ULK1, a key regulator of autophagy, in response to starvation-inactivated MTOR signaling . This connection may represent a novel mechanism by which cancer cells adapt to nutrient deprivation.

• Cancer stemness: Elevated SH3PXD2A expression has been associated with cancer stem cell-like properties in multiple tumor types, suggesting a role in tumor initiation and therapy resistance.

• Metabolic reprogramming: Emerging evidence indicates SH3PXD2A may influence cancer cell metabolism, potentially through interactions with metabolic enzymes or signaling pathways.

• Tumor microenvironment modulation: SH3PXD2A may contribute to remodeling the tumor microenvironment beyond ECM degradation, potentially influencing immune cell recruitment and function.

These expanding roles suggest SH3PXD2A functions as a multifaceted regulator in cancer progression, making it an attractive target for comprehensive cancer therapeutic strategies .

How does SH3PXD2A expression correlate with clinical outcomes in different cancer types?

Analysis of clinical data reveals significant correlations between SH3PXD2A expression and patient outcomes across multiple cancer types:

These findings highlight the clinical relevance of monitoring SH3PXD2A expression in cancer patients and suggest its potential utility in clinical decision-making for treatment strategies.

What novel therapeutic approaches target SH3PXD2A or its signaling pathways?

Emerging therapeutic strategies targeting SH3PXD2A and its associated pathways include:

• Direct SH3PXD2A inhibition:

  • Small molecule inhibitors targeting the PX domain to disrupt phospholipid binding

  • Peptide-based inhibitors disrupting key protein-protein interactions mediated by SH3 domains

• Upstream regulation targeting:

  • SRC kinase inhibitors to prevent SH3PXD2A phosphorylation and activation

  • Modulators of cytokine signaling that trigger SH3PXD2A activation

• RNA-based therapeutics:

  • siRNAs or antisense oligonucleotides targeting SH3PXD2A

  • LncRNA-targeted therapies against SH3PXD2A-AS1, which has been shown to promote NSCLC growth

• Combination approaches:

  • Combining SH3PXD2A inhibition with MMP inhibitors

  • Dual targeting of SH3PXD2A and DHX9, as this interaction enhances FOXM1 expression

These therapeutic strategies are in various stages of development, from preclinical to early clinical investigation, and represent promising approaches for cancers where SH3PXD2A plays a significant role in progression and metastasis.

What are the recommended positive and negative controls for SH3PXD2A antibody validation?

For robust validation of SH3PXD2A antibodies, the following controls are recommended:

Positive Controls:

• Cell lines: HeLa and HepG2 cells have been validated for Western blot applications

• Tissues: Human breast cancer tissue has been validated for IHC applications

• Overexpression systems: Cells transfected with SH3PXD2A expression vectors

Negative Controls:

• Knockdown/knockout validation:

  • siRNA or shRNA-mediated knockdown of SH3PXD2A

  • CRISPR/Cas9-mediated knockout cells

• Peptide competition assays: Pre-incubation of antibody with immunizing peptide

• Isotype controls: Rabbit IgG at equivalent concentration for rabbit polyclonal antibodies

Additional Technical Controls:

• Multiple antibodies approach: Using antibodies targeting different epitopes of SH3PXD2A

• Species validation: Comparing reactivity across human, mouse, and rat samples when appropriate

• Expected molecular weight verification: Confirming band appears at 140-150 kDa despite calculated MW of 125 kDa

What are the key technical specifications to consider when selecting an SH3PXD2A antibody?

When selecting an SH3PXD2A antibody for research, consider these critical technical specifications:

• Epitope location and sequence:

  • Antibodies targeting different regions may yield different results

  • Consider the immunogen sequence (e.g., amino acids 806-1105 of human SH3PXD2A )

  • For detecting specific isoforms or phosphorylated forms, select antibodies with appropriate epitope recognition

• Validation status for specific applications:

  • Verify antibody has been validated for your intended application (WB, IHC, IF/ICC, CoIP)

  • Review published literature citing the specific antibody

• Species reactivity:

  • Confirm reactivity with your species of interest (human, mouse, rat)

  • Consider cross-reactivity with other species if relevant

• Antibody format and characteristics:

  • Host species and isotype (typically rabbit IgG for many SH3PXD2A antibodies)

  • Clonality (monoclonal vs. polyclonal)

  • Concentration and formulation

  • Storage requirements and stability

• Quality control metrics:

  • Batch-to-batch consistency

  • Purity assessment

  • Specificity validation methods

• Additional considerations:

  • Recommended dilutions for specific applications

  • Buffer compatibility

  • Secondary antibody requirements

Creating a comparison table of commercially available antibodies against these criteria can facilitate selection of the most appropriate antibody for your specific research needs.

How can researchers effectively collaborate and share SH3PXD2A research findings?

To enhance collaboration and knowledge sharing in SH3PXD2A research:

• Standardized reporting:

  • Thoroughly document antibody information (catalog number, lot, dilution, incubation conditions)

  • Report all experimental conditions in detail

  • Include all controls used for validation

  • Present both positive and negative results

• Data repositories and databases:

  • Deposit raw data in appropriate repositories

  • Contribute validated antibody information to antibody validation databases

  • Share protocols on platforms like protocols.io

• Open science practices:

  • Consider preprint servers for rapid dissemination

  • Participate in collaborative research networks

  • Share materials through repositories or direct collaborations

• Technical forums and communities:

  • Engage in research-specific online communities

  • Participate in specialized conferences and workshops

  • Contribute to method-focused publications

• Cross-disciplinary engagement:

  • Connect with researchers studying related molecules (e.g., SH3PXD2A-AS1, DHX9, FOXM1)

  • Engage with clinical researchers to translate findings

  • Collaborate with computational biologists for systems-level analyses