SH3BGRL3 Antibody

What is SH3BGRL3 and what are its key structural features?

SH3BGRL3 is a 10.5kDa protein with an isoelectric point of 5.0 that belongs to the thioredoxin-like protein family. The SH3BGRL3 gene is located on chromosome 1p34.3-35 and encodes a 93 amino acid protein. While showing significant similarity to glutaredoxin 1 of E. coli, SH3BGRL3 lacks the canonical sequence of redox active sites and is devoid of enzymatic activity. Unlike other members of its family, it does not have a canonical SH3 binding domain, displaying only a PPQIV sequence instead of the SH3 consensus sequence PXXPQ(L/I)(Y/F) .

The three-dimensional structure of SH3BGRL3 has been resolved by X-ray crystallography and nuclear magnetic resonance (NMR), revealing important insights about its potential functional domains .

What methodologies are most effective for detecting SH3BGRL3 in different experimental contexts?

SH3BGRL3 can be detected using multiple methodologies depending on your experimental goals:

For protein expression analysis:

• Western Blotting: Most commercial SH3BGRL3 antibodies work at dilutions of 1:500-1:2000. The observed molecular weight is typically 10 kDa .

• Immunohistochemistry: Recommended dilutions range from 1:400-1:1600, with antigen retrieval preferably performed with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 .

For tissue/cellular localization:

• Confocal microscopy with immunofluorescence has successfully demonstrated SH3BGRL3 co-localization with other proteins like Myosin 1c .

For transcriptomic analysis:

• RT-qPCR has been effectively used to measure SH3BGRL3 mRNA expression in clinical samples, as demonstrated in studies of acute myeloid leukemia .

For clinical specimens:

• Tissue microarray (TMA) and immunohistochemistry (IHC) have been successfully employed to assess SH3BGRL3 protein expression in gastric cancer and correlate it with clinicopathological parameters .

How can I validate SH3BGRL3 antibody specificity for my research?

A multi-step validation approach is recommended:

• Protein knockdown validation: Use siRNA-mediated knockdown of SH3BGRL3 followed by Western blot to confirm specificity. Research shows that transfection with specific SH3BGRL3 siRNAs effectively decreases protein expression in cancer cell lines such as MDA-MB-231 .

• Overexpression validation: Transfect cells with SH3BGRL3 expression vectors and confirm increased signal intensity in Western blot or immunofluorescence.

• Multiple antibody verification: When possible, use antibodies targeting different epitopes of SH3BGRL3 and compare results.

• Cross-reactivity assessment: Test the antibody on samples from multiple species if conducting comparative studies. Commercial antibodies show reactivity with human, mouse, and rat samples .

• Control tissues/cells: Include known positive and negative controls based on documented SH3BGRL3 expression patterns.

How can I effectively study SH3BGRL3's role in cancer progression?

SH3BGRL3 has been implicated in multiple cancer types, requiring specific methodological approaches:

For cellular migration studies:

• Modulate SH3BGRL3 expression through siRNA knockdown or overexpression in cancer cell lines.

• Utilize Boyden chamber assays to quantify migration capacity.

• Compare control cells with SH3BGRL3-modulated cells.

Research has demonstrated that SH3BGRL3 downregulation via siRNA results in significant decrease of migration capacity in MDA-MB-231 cells. Conversely, overexpression of SH3BGRL3 significantly increases cell migration ability .

For clinical significance assessment:

• Analyze SH3BGRL3 expression in tumor tissues compared to adjacent normal tissues.

• Correlate expression levels with clinicopathological parameters and patient outcomes.

• Consider Kaplan-Meier survival analysis to evaluate prognostic value.

What are the optimal protocols for studying SH3BGRL3 interaction with Myosin 1c?

SH3BGRL3 has been demonstrated to interact with Myosin 1c (Myo1c) in a calcium-dependent manner. To study this interaction:

Co-immunoprecipitation approach:

• Engineer cells to express exogenous FLAG-SH3BGRL3

• Prepare cell lysates with proper calcium concentration maintenance

• Use anti-FLAG-coupled resin for immunoprecipitation

• Analyze bound proteins by SDS-PAGE followed by Western blotting using anti-Myo1c antibodies

Confocal microscopy co-localization:

• Use cells expressing both proteins (naturally or through transfection)

• Employ antibodies against both SH3BGRL3 and Myo1c

• Focus on membrane structures, particularly membrane ruffles where co-localization is most evident

Research has shown that SH3BGRL3 specifically recognizes Myo1c on its IQ-bearing neck region, and that this interaction is calcium-dependent .

How can I investigate the relationships between SH3BGRL3 expression and immune cell infiltration in tumors?

Recent research has revealed potential connections between SH3BGRL3 and tumor immune microenvironment:

• Apply ssGSEA (single-sample Gene Set Enrichment Analysis) and CIBERSORT to quantify relative infiltration levels of immune cell subsets in relation to SH3BGRL3 expression.

• Analyze gene expression data using TIMER and TISIDB online tools to assess relationships between SH3BGRL3 expression and immune infiltration.

• Validate findings using immunohistochemistry on patient samples with antibodies targeting both SH3BGRL3 and immune cell markers.

Studies in acute myeloid leukemia have shown that B cells, naïve plasma cells, T cells, CD4 memory resting cells, and resting NK cells were significantly downregulated in samples with high SH3BGRL3 expression, suggesting its involvement in tumor immune microenvironment modulation .

What techniques can accurately distinguish between SH3BGRL3 and its circular RNA form (circSH3BGRL3)?

Both SH3BGRL3 and its circular RNA form (circSH3BGRL3) have been implicated in cancer progression, requiring specific techniques to distinguish between them:

For detection of circular RNA:

• Design divergent primers that can only amplify circular products

• Use RNase R treatment to enrich for circular RNAs (circular RNAs are resistant to exonuclease digestion)

• Confirm circularity by Sanger sequencing across the back-splice junction

For stability analysis:

• Treat cells with actinomycin D (transcription inhibitor) and harvest cells at different time points

• Extract RNA and perform qRT-PCR for both circSH3BGRL3 and SH3BGRL3 mRNA

• Compare degradation rates (circular RNAs typically show higher stability)

Research has demonstrated that circSH3BGRL3 is a stable circRNA expressed in human AML cells. Divergent primers amplified circular products in cDNA but not in genomic DNA, whereas convergent primers amplified linear products in both cDNA and genomic DNA .

What explains the contradictory findings regarding SH3BGRL3's interaction with ErbB2/EGFR?

Research on SH3BGRL3's interaction with receptor tyrosine kinases has yielded contradictory results that require careful interpretation:

• Confocal microscopy shows co-localization of SH3BGRL3 and ErbB2 primarily at membrane structures .

• Co-immunoprecipitation experiments fail to confirm direct binding between SH3BGRL3 and ErbB2 .

• Structural analysis indicates SH3BGRL3 lacks the SH2 domain required for direct docking to phosphorylated tyrosine residues on receptors .

These contradictions can be resolved by understanding that SH3BGRL3 may interact with EGFR family members indirectly through adaptor proteins like Myosin 1c, which itself localizes to membrane structures. The experimental approach matters significantly - while co-localization in microscopy doesn't necessarily indicate direct binding, it may suggest functional proximity in cellular contexts.

What controls are essential when studying SH3BGRL3 expression in cancer tissues?

When investigating SH3BGRL3 expression in cancer tissues, include these critical controls:

For immunohistochemistry:

• Tissue controls: Always include adjacent normal tissue alongside tumor samples. Studies show SH3BGRL3 exhibits aberrant expression in tumor tissues compared to adjacent normal tissues .

• Antibody controls: Include isotype control antibodies to assess non-specific binding.

• Expression validation: When possible, validate protein expression using multiple techniques (IHC, Western blot, RT-qPCR).

For prognostic studies:

• Multivariate analysis: Control for known prognostic factors when assessing SH3BGRL3's independent prognostic value.

• Cancer subtype stratification: In gastric cancer, SH3BGRL3's prognostic value differs between Epstein-Barr virus-negative and positive subtypes .

Table compiled from data reported in clinical studies of gastric cancer patients

How can I optimize SH3BGRL3 detection in different sample preparations?

Specific optimization strategies are needed for different sample types:

For Western blotting:

• Use recommended dilutions of 1:500-1:2000 for commercial antibodies

• Expect to observe SH3BGRL3 at approximately 10 kDa

• Include proper loading controls appropriate for your experimental context

For immunohistochemistry on FFPE tissues:

• Optimal antigen retrieval is critical - use TE buffer pH 9.0 as first choice

• Alternative antigen retrieval with citrate buffer pH 6.0 if needed

• Use recommended antibody dilutions of 1:400-1:1600

For RT-qPCR detection:

• Design primers spanning exon junctions to avoid genomic DNA amplification

• Validate primer specificity through melt curve analysis and sequencing

• Use appropriate reference genes that show stability in your experimental conditions

What is the current evidence linking SH3BGRL3 to cell migration and metastasis?

Multiple lines of evidence connect SH3BGRL3 to cell migration and potential metastasis:

• Molecular interaction mechanism: SH3BGRL3 binds to Myosin 1c, a motor protein involved in cytoskeleton dynamics and membrane tension .

• Functional studies:

  • SH3BGRL3 knockdown via siRNA significantly decreases migration capacity in MDA-MB-231 cells

  • SH3BGRL3 overexpression increases cell motility in migration assays

  • These effects have been quantified using Boyden chamber assays

• Clinical correlations: Higher SH3BGRL3 expression correlates with tumor progression parameters including increased TNM staging, tumor budding, and perineural invasion in gastric cancer .

This evidence suggests SH3BGRL3 may promote cancer progression by enhancing cellular motility through its interaction with cytoskeletal components, particularly Myosin 1c.

How does SH3BGRL3 expression correlate with clinical outcomes in different cancer types?

SH3BGRL3 expression shows significant associations with clinical outcomes across multiple cancer types:

In Acute Myeloid Leukemia (AML):

In Gastric Cancer:

In Other Cancers:

• Increased SH3BGRL3 expression has been documented in lung adenocarcinoma, bladder cancer, and kidney cancer

• In no muscle-invasive bladder cancer, high SH3BGRL3 expression significantly correlates with increased risk of progression

What methodological approaches can elucidate SH3BGRL3's role in drug resistance mechanisms?

Recent research reveals SH3BGRL3's potential involvement in chemoresistance, particularly in AML:

• Gene expression profiling:

  • Analyze differentially expressed genes between parental AML cell lines and drug-resistant derivatives

  • SH3BGRL3 was identified as one of the dysregulated mRNA molecules between parental HL-60 cells and daunorubicin-resistant cells (HRD)

  • The expression of SH3BGRL3 in daunorubicin-resistant AML cells significantly increased relative to normal AML cells

• Functional validation:

  • Determine IC50 values of chemotherapeutic agents in cell lines with different SH3BGRL3 expression levels

  • Silence circSH3BGRL3 using siRNA approaches and evaluate changes in drug sensitivity

  • Studies show that silencing of circSH3BGRL3 affects resistance of AML cells to daunorubicin, with more obvious proliferation arrest when treated with both circSH3BGRL3 knockdown and daunorubicin

• Mechanism exploration:

  • Gene functional enrichment analysis reveals that differentially expressed genes related to SH3BGRL3 are mainly enriched in ATP metabolism, ATP synthesis, oxidative phosphorylation and electron transport chain in gastric cancer

  • These metabolic pathways are known to influence drug resistance mechanisms

What are the most promising directions for studying SH3BGRL3's role in redox regulation?

Despite SH3BGRL3's structural similarity to glutaredoxins, its precise role in redox regulation remains incompletely understood:

These approaches could reveal how SH3BGRL3 contributes to cancer cell survival under oxidative stress conditions, potentially opening new therapeutic avenues.