SH2D4A Antibody

What is SH2D4A and what cellular functions does it regulate?

SH2D4A (SH2 Domain-containing Protein 4A) is a novel signaling adapter protein involved in multiple cellular processes. Research indicates that SH2D4A plays a crucial role in mitotic progression by promoting centrosome maturation and spindle microtubule formation. It contributes significantly to chromosome alignment during the prometaphase stage of mitosis . SH2D4A enhances microtubule nucleation and polymerization, supporting kinetochore-microtubule attachment through the recruitment of pericentriolar material (PCM) components such as Cep192, γ-tubulin, and active PLK1 to centrosomes .

Beyond its mitotic functions, SH2D4A has been identified as a potential tumor suppressor gene located on chromosome 8p. Studies demonstrate that it can inhibit Signal Transducer and Activator of Transcription 3 (STAT3) signaling by directly interacting with STAT3 and retaining it in the cytoplasm, thereby preventing its transcriptional activity . This inhibitory effect on STAT3 signaling suggests SH2D4A may have important implications in cancer development and progression.

How is SH2D4A structurally characterized and what domains does it contain?

SH2D4A possesses a single SH2 (Src Homology 2) domain located at the C-terminal end of the protein. This SH2 domain shares significant homology with other adapter proteins, specifically 66% homology with the TSAd (T cell-specific adapter protein) SH2 domain and 71% homology with the ALX (adapter protein in lymphocytes of unknown function) SH2 domain in mice . The SH2D4A SH2 domain is classified as a type I SH2 domain and contains conserved residues predicted to form contacts with phosphorylated tyrosine residues in target proteins .

Beyond the SH2 domain, SH2D4A features conserved tyrosine residues in consensus phosphorylation motifs and a proline-rich region. These structural features potentially enable interaction with SH2 and SH3 domains of other signaling molecules, suggesting a scaffold function for protein-protein interactions . The complete protein has a predicted molecular mass of approximately 52.7 kDa, which corresponds to the ~52 kDa band observed in Western blot analyses .

What is the expression profile of SH2D4A across different tissues and cell types?

SH2D4A exhibits a ubiquitous expression pattern at varying levels across multiple tissues and cell types. Analysis of expressed sequence tag databases and public microarray data indicates that SH2D4A is expressed at low levels in diverse tissues . Western blot analyses have confirmed SH2D4A protein expression in immune cells, including human peripheral blood mononuclear cells (PBMCs) .

In mouse models, SH2D4A expression has been detected in CD4+ T cells, CD8+ T cells, B cells, bone marrow-derived macrophages, and dendritic cells . Additionally, SH2D4A expression has been reported in various cancer cell lines, including hepatocellular carcinoma (HCC) cell lines and SV40 large T antigen transformed normal liver THLE-2 cells, though the expression levels vary considerably between different cell lines .

In colorectal cancer research, SH2D4A has been detected in normal colorectal tissue and adenomas, with certain colorectal carcinomas showing decreased or absent expression, suggesting a potential role in tumor progression .

What are the validated applications for SH2D4A antibodies in research?

SH2D4A antibodies have been successfully employed in several research applications with specific validation parameters:

Western Blotting: SH2D4A antibodies have been extensively validated for Western blot applications. Multiple studies have utilized anti-SH2D4A antibodies to detect the protein at approximately 52 kDa in various cell lysates . The antibodies work effectively with standard protein extraction protocols and conventional Western blotting techniques.

Immunohistochemistry (IHC): Anti-SH2D4A antibodies have been validated for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues. Studies have successfully used SH2D4A antibodies to evaluate cytoplasmic immunoreactivity in tumor cells and normal tissues, enabling classification of samples as SH2D4A-positive or SH2D4A-negative .

Co-immunoprecipitation: SH2D4A antibodies have been employed in co-immunoprecipitation assays to investigate protein-protein interactions, particularly the interaction between SH2D4A and STAT3, both in situ and in vitro .

What are the recommended protocols for SH2D4A antibody use in Western blotting?

For optimal Western blot results with SH2D4A antibodies, the following protocol parameters are recommended based on published research:

Sample Preparation:

• Extract proteins using standard lysis buffers compatible with phosphoprotein preservation

• Include protease and phosphatase inhibitors in extraction buffers

Western Blotting Conditions:

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

• Block membranes with 5% nonfat dried skimmed milk in TBS-T

• Incubate with primary SH2D4A antibody (recommended dilution 1:1000 for Sigma-Aldrich HPA001919)

• Wash with TBS-T

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using ECL detection reagents

Antibody Validation Controls:

• Include positive control lysates from cells known to express SH2D4A

• Include negative control from SH2D4A-knockdown or knockout cells

• Use β-actin (1:5000 dilution) as a loading control

How should researchers optimize SH2D4A antibody protocols for immunohistochemistry?

Based on published research utilizing SH2D4A antibodies for immunohistochemistry, the following optimization strategies are recommended:

Tissue Preparation:

• Formalin fixation followed by paraffin embedding is suitable for SH2D4A detection

• Consider heat-induced epitope retrieval methods to ensure optimal antibody binding

Staining Protocol:

• Deparaffinize and rehydrate tissue sections

• Perform antigen retrieval (specific conditions may need optimization)

• Block endogenous peroxidase activity

• Incubate with primary SH2D4A antibody (optimal dilution should be determined empirically)

• Apply appropriate detection system (e.g., polymer-based detection systems)

• Counterstain, dehydrate, and mount

Evaluation Criteria:

• Assess cytoplasmic SH2D4A immunoreactivity in target cells

• Classify results as SH2D4A-positive or SH2D4A-negative, where negative is defined as no staining of SH2D4A in target cells

• For quantitative assessment, evaluate staining in multiple independent areas (at least four) at appropriate magnification (e.g., ×400)

How does SH2D4A contribute to mitotic progression through centrosome maturation?

SH2D4A plays a critical role in promoting centrosome maturation during mitosis through several interrelated mechanisms:

PCM Recruitment Pathway: SH2D4A facilitates the recruitment of pericentriolar material (PCM) components to centrosomes, which is essential for centrosome maturation. Specifically, SH2D4A promotes the centrosomal recruitment of key proteins including Cep192, γ-tubulin, active PLK1 (Polo-like kinase 1), and active Aurora A kinase .

Phosphatase Regulation: SH2D4A contributes to centrosome maturation by attenuating protein phosphatase 1 (PP1) activity. Research has demonstrated that SH2D4A binds to PP1α/β during mitosis, thereby inhibiting PP1 phosphatases. This inhibition enhances the phosphorylation-dependent maturation of centrosomes, as evidenced by experiments showing that treatment with the PP1/PP2A inhibitor calyculin A can rescue defects in PLK1 activation and microtubule nucleation caused by SH2D4A knockdown .

Impact on Spindle Assembly: Through promoting centrosome maturation, SH2D4A supports robust spindle microtubule formation. Time-lapse imaging analysis of SH2D4A-knockdown cells revealed prolonged duration of prophase/prometaphase, with the most significant effect observed in chromosome alignment . Cold treatment assays demonstrated that SH2D4A knockdown reduced cold-stable microtubules, indicating impaired kinetochore-microtubule attachments .

Quantitative Effects on Mitotic Timing: SH2D4A depletion significantly prolongs mitotic progression. In control cells, the average time from mitotic entry to exit was 70.5 minutes, while SH2D4A-knockdown cells required 88.6 minutes (siSH2D4A #1) and 87.5 minutes (siSH2D4A #2) . This delay was primarily attributable to extended prometaphase duration.

What is the relationship between SH2D4A expression and STAT3 signaling pathways?

SH2D4A functions as a negative regulator of STAT3 signaling through direct and indirect mechanisms:

Direct STAT3 Inhibition: Co-immunoprecipitation studies, both in situ and in vitro, have demonstrated that SH2D4A directly interacts with STAT3 . This interaction results in cytoplasmic retention of STAT3, preventing its nuclear translocation and subsequent transcriptional activity . This provides a molecular mechanism for SH2D4A's inhibitory effect on STAT3-mediated gene expression.

Impact on IL-6 Signaling: Overexpression of SH2D4A in hepatocellular carcinoma (HCC) cells leads to decreased expression of IL-6 target genes, which are typically regulated by STAT3 signaling . This suggests that SH2D4A can attenuate IL-6/STAT3 signaling pathways that are frequently hyperactivated in various cancers.

Cooperative Tumor Suppression: SH2D4A appears to function cooperatively with another chromosome 8p gene, SORBS3, to inhibit STAT3 signaling. While SH2D4A directly interacts with STAT3, SORBS3 co-activates estrogen receptor α (ERα) signaling, which indirectly represses STAT3 activity . This coordinated activity provides a mechanistic explanation for their linked tumor suppressor functions.

Immune Microenvironment Influence: In human HCC tissues, SH2D4A expression positively correlates with infiltrating regulatory and cytotoxic T cell populations . Similarly, in colorectal cancer, loss of SH2D4A expression correlates with scarce T cell infiltration . These findings suggest that SH2D4A may influence tumor immunogenicity through its effects on STAT3 signaling, potentially explaining the "immune cold" phenotype observed in tumors with SH2D4A loss.

How does SH2D4A downregulation impact chromosome 8p-associated tumorigenesis?

SH2D4A downregulation contributes to chromosome 8p-associated tumorigenesis through multiple mechanisms:

Progressive Loss During Tumor Evolution: Analysis of the adenoma-carcinoma sequence in microsatellite stable/chromosomal instability (MSS/CIN) colorectal cancers revealed that SH2D4A is part of a set of 11 genes on chromosome 8p that are progressively downregulated during tumor evolution . All adenomas were found to be SH2D4A-positive by immunohistochemistry, while a subset (5.3%) of colorectal carcinomas showed complete loss of SH2D4A expression .

Prognostic Significance: Loss of SH2D4A expression correlates with poor prognosis in colorectal cancer patients. This negative prognostic impact was validated in independent cohorts, suggesting that SH2D4A downregulation contributes to more aggressive disease phenotypes .

Immunological Impact: SH2D4A loss is associated with reduced T cell infiltration in tumors, contributing to an "immune cold" tumor microenvironment . This immunological effect may explain, in part, the poorer outcomes observed in patients with SH2D4A-negative tumors, as immune surveillance is compromised.

Cooperative Gene Effects: Evidence suggests that SH2D4A downregulation likely acts in concert with other chromosome 8p genes to promote tumorigenesis. The combined downregulation of multiple tumor suppressor genes on chromosome 8p appears to have a cooperative effect that is greater than the loss of any single gene . This cooperative model explains why chromosome 8p deletions are frequent events in multiple cancer types.

What controls should be included when using SH2D4A antibodies in experimental studies?

To ensure reliable and interpretable results when using SH2D4A antibodies, researchers should include the following controls:

Positive Controls:

• Cell lines with confirmed SH2D4A expression (e.g., certain HCC cell lines, normal human PBMCs)

• Normal tissues known to express SH2D4A (e.g., colorectal adenoma tissue)

• Recombinant SH2D4A protein for Western blot standardization

Negative Controls:

• SH2D4A knockdown cells using validated siRNAs targeting SH2D4A (e.g., siSH2D4A #1 and siSH2D4A #2 as described in the literature)

• SH2D4A knockout cells or tissues, ideally generated using CRISPR-Cas9 or similar technology

• For IHC, include secondary antibody-only controls to assess background staining

Specificity Validation:

• Rescue experiments using ectopic expression of SH2D4A in knockdown cells

• SH2D4A inducible expression systems, such as Doxycycline-inducible HA-tagged SH2D4A cell lines (A549/HA-SH2D4A or RPE-1/HA-SH2D4A)

• Antibody pre-absorption with immunizing peptide for Western blot or IHC

Loading/Staining Controls:

• For Western blotting, use housekeeping proteins such as β-actin (1:5000 dilution) or PCNA (1:5000 dilution)

• For IHC, include evaluation of serial sections with established markers

How can researchers effectively generate and validate SH2D4A knockdown models?

Based on published research, the following approaches have been validated for generating and confirming SH2D4A knockdown models:

siRNA-Mediated Knockdown:

• Use multiple siRNA sequences targeting different regions of SH2D4A mRNA to control for off-target effects

  • siSH2D4A #1: targeting coding region

  • siSH2D4A #2: targeting 3'-untranslated region (3'UTR)

• Transfect target cells using standard lipid-based transfection reagents

• Allow 48-72 hours for effective protein depletion

• Confirm knockdown by Western blot using validated anti-SH2D4A antibodies

Rescue Experiments to Validate Specificity:

• Establish inducible expression system for SH2D4A (e.g., Doxycycline-inducible)

• Target endogenous SH2D4A with siRNA directed against 3'UTR (siSH2D4A #2)

• Induce expression of exogenous SH2D4A (lacking the 3'UTR) using Doxycycline

• Confirm rescue of phenotype to validate specificity of knockdown effects

Phenotypic Validation:

• Assess mitotic progression using synchronization with CDK1 inhibitor RO-3306

• Perform time-lapse imaging to evaluate specific mitotic phase delays

• Examine microtubule nucleation using cold treatment assay and microtubule regrowth assay

• Evaluate centrosome maturation by immunofluorescence for PCM markers

How can researchers troubleshoot negative or weak SH2D4A immunostaining results?

When encountering weak or negative SH2D4A immunostaining results, researchers should consider the following troubleshooting approaches:

Antibody-Related Factors:

• Verify antibody specificity using Western blot on positive control samples

• Optimize antibody concentration (test a range of dilutions)

• Consider alternative SH2D4A antibodies targeting different epitopes

• Ensure proper antibody storage conditions to prevent degradation

Sample Preparation Issues:

• Evaluate fixation conditions (overfixation can mask epitopes)

• Optimize antigen retrieval methods:

  • Test different pH buffer systems (citrate pH 6.0 vs. EDTA pH 9.0)

  • Adjust retrieval time and temperature

• Ensure tissue processing maintains protein integrity

Detection System Optimization:

• Consider more sensitive detection systems (e.g., polymer-based vs. ABC method)

• Increase incubation time with primary antibody (overnight at 4°C)

• Evaluate signal amplification methods for low-expressing samples

Biological Considerations:

• Verify SH2D4A expression levels in the specimen type by complementary methods

• Consider that certain tumors genuinely lack SH2D4A expression (5.3% of colorectal carcinomas are SH2D4A-negative)

• Examine chromosome 8p status if possible, as deletions may explain absence of SH2D4A

How is SH2D4A expression altered in different cancer types and what are the implications?

SH2D4A expression shows distinctive patterns of alteration across cancer types, with significant functional and clinical implications:

Colorectal Cancer (CRC):

• SH2D4A is part of a set of 11 genes progressively downregulated during the adenoma-carcinoma sequence in microsatellite stable/chromosomal instability (MSS/CIN) colorectal cancers

• While all adenomas maintain SH2D4A expression, approximately 5.3% of colorectal carcinomas completely lose SH2D4A immunoreactivity

• SH2D4A loss in CRC correlates with poor prognosis and reduced T-cell infiltration in the tumor microenvironment

• Loss typically occurs through chromosome 8p deletions rather than mutations in the SH2D4A gene itself

Hepatocellular Carcinoma (HCC):

• SH2D4A functions as a tumor suppressor in HCC by inhibiting STAT3 signaling

• SH2D4A works cooperatively with another chromosome 8p gene, SORBS3, to suppress HCC progression

• In HCC tissues, SH2D4A expression positively correlates with infiltrating regulatory and cytotoxic T cell populations, suggesting it influences the immune microenvironment

Clinical Implications:

• SH2D4A status may serve as a prognostic biomarker, particularly in colorectal cancer

• The association between SH2D4A loss and reduced immune cell infiltration suggests potential implications for immunotherapy response prediction

• The coordinated loss of multiple chromosome 8p genes, including SH2D4A, may represent a distinct molecular subtype of cancer with unique therapeutic vulnerabilities

What methodologies are recommended for studying SH2D4A's role in tumor immunology?

Based on published research, the following methodologies are recommended for investigating SH2D4A's role in tumor immunology:

Tissue-Based Immune Profiling:

• Multiplex immunohistochemistry to simultaneously evaluate SH2D4A and immune cell markers (CD4, CD8, Foxp3)

• Quantitative assessment of immune infiltration:

  • Evaluate invasive front region in four independent areas

  • Count stained lymphocytes at 400× magnification

  • For Foxp3+ regulatory T cells, select four independent hotspot areas for counting

• Correlate SH2D4A expression with immune infiltration patterns

Transcriptomic Analysis:

• Apply immune gene signatures (e.g., 141-immune gene signature according to ESTIMATE) to assess immune infiltration in SH2D4A-high vs. SH2D4A-low tumors

• Use RNA sequencing or microarray data to identify immunological pathways differentially regulated in relation to SH2D4A status

• Validate key findings using qRT-PCR in cell line models with SH2D4A manipulation

Functional Studies:

• Co-culture experiments with immune cells and tumor cells with/without SH2D4A expression

• Cytokine profiling in SH2D4A-manipulated tumor cells to assess immunomodulatory secretome

• Investigate effects of SH2D4A expression on response to immune checkpoint inhibitors in preclinical models

In Vivo Models:

• Compare tumor growth and immune infiltration in immunocompetent mice using SH2D4A-expressing vs. SH2D4A-knockdown tumor cells

• Assess response to immunotherapies in relation to SH2D4A status

• Consider humanized mouse models for more translational relevance

What are the emerging research questions regarding SH2D4A function and regulation?

Several critical research questions about SH2D4A remain to be fully elucidated:

• Upstream Regulation: What regulates SH2D4A expression and activity in normal and disease states? Are there specific transcription factors or epigenetic mechanisms controlling SH2D4A expression?

• Post-translational Modifications: How do post-translational modifications affect SH2D4A function? The protein contains conserved tyrosine residues in consensus phosphorylation motifs , but their phosphorylation status and functional significance remain largely unexplored.

• Protein Interaction Network: Beyond STAT3 and PP1 phosphatases, what other proteins interact with SH2D4A? A comprehensive interactome analysis would provide valuable insights into its various cellular functions.

• Tissue-Specific Functions: Does SH2D4A have tissue-specific functions beyond its roles in mitosis and STAT3 regulation? Its ubiquitous expression pattern suggests potential diverse functions depending on cellular context.

• Therapeutic Targeting: Can restoration of SH2D4A function or targeting its downstream pathways provide therapeutic benefit in cancers with chromosome 8p deletions?

• Biomarker Development: Can SH2D4A status be developed into a clinically useful biomarker for cancer prognosis or treatment response prediction, particularly for immunotherapies?

Addressing these questions will require integrated approaches combining structural biology, proteomics, genomics, and translational research to fully understand SH2D4A's biological significance and therapeutic potential.