HS2ST1 Antibody

What is HS2ST1 and what cellular functions does it regulate?

HS2ST1 (heparan sulfate 2-O-sulfotransferase 1) is an enzyme involved in the biosynthesis of heparan sulfate proteoglycans (HSPGs). It specifically mediates 2-O-sulfation of heparan sulfate. HS2ST1 plays important roles in regulating cell migration, invasion, and differentiation through modulation of multiple signaling pathways. In breast cancer cells, HS2ST1 influences cell viability, adhesion to fibronectin and laminin, and invasive behavior by altering growth factor binding to cell surface receptors . The calculated molecular weight of HS2ST1 is 27 kDa (229 amino acids), matching its observed molecular weight in experimental settings . HS2ST1 expression is dysregulated in several tumor entities, suggesting its role in tumor progression, and has been associated with cancer stem cell signatures in breast cancer cells .

What applications are validated for HS2ST1 antibodies?

HS2ST1 antibodies have been validated for multiple research applications:

Positive western blot detection has been confirmed in human liver tissue, human brain tissue, human heart tissue, and PC-3 cells . For immunohistochemistry applications, human lymphoma tissue has been validated as positive control material . The antibody has also been cited in publications for IHC applications, particularly in cancer research .

How should HS2ST1 antibodies be stored to maintain optimal activity?

HS2ST1 antibodies should be stored at -20°C to maintain optimal activity. The antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) . Under these storage conditions, the antibody remains stable for one year after shipment . Most commercial preparations indicate that aliquoting is unnecessary for -20°C storage, which simplifies laboratory handling . Some preparations (20μl sizes) contain 0.1% BSA, which helps maintain antibody stability . For shipping purposes, the antibody is typically transported on wet ice .

What antigen retrieval methods are recommended for HS2ST1 immunohistochemistry?

For optimal immunohistochemical detection of HS2ST1, specific antigen retrieval methods are recommended:

• Primary recommendation: Antigen retrieval with TE buffer pH 9.0 is suggested as the optimal method for unmasking HS2ST1 epitopes in formalin-fixed, paraffin-embedded tissues .

• Alternative method: Citrate buffer pH 6.0 can be used as an alternative antigen retrieval approach if the primary method is not yielding satisfactory results .

The choice of antigen retrieval method can significantly impact the sensitivity and specificity of HS2ST1 detection in tissue samples. Researchers should validate these conditions in their specific tissue types, as retrieval efficiency may vary depending on tissue fixation duration, embedding protocols, and the age of tissue blocks .

How should appropriate controls be selected for HS2ST1 expression studies?

When designing HS2ST1 expression studies, proper selection of controls is critical:

For positive tissue controls:

• Human lymphoma tissue has been validated for IHC applications

• Human liver, brain, and heart tissues have been confirmed for western blot applications

• PC-3 cells (prostate cancer cell line) can serve as positive cell line controls

For experimental controls in gene modulation studies:

• Vector control cells should be used as baseline controls when performing overexpression studies

• Scrambled siRNA controls should be included for knockdown experiments

• When studying cancer stem cell phenotypes, appropriate parental cell lines (e.g., MDA-MB-231 and MCF-7 for breast cancer studies) should be selected based on their basal expression of HS2ST1

Expression of housekeeping genes should be monitored to normalize HS2ST1 expression levels, and qPCR validation of expression changes should precede functional studies. For instance, qPCR revealed that HS2ST1 overexpression led to a 25-37-fold increase in HS2ST1 mRNA expression in transfected cells compared to controls .

How does HS2ST1 expression modulate cancer cell invasion and migration?

HS2ST1 has significant and sometimes contradictory effects on cancer cell invasion and migration:

In breast cancer cells, HS2ST1 overexpression inhibits Matrigel invasion, while its knockdown reverses this phenotype . This inhibitory effect on invasion is associated with:

• Altered cell adhesion to fibronectin and laminin matrices

• Reduced signaling through multiple pathways, particularly the MAPK pathway

• Changed binding of fibroblast growth factor 2 (FGF-2) to cell surfaces

• Altered expression of key proteins including epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4

The mechanism appears to involve HS2ST1-mediated structural changes in heparan sulfate, which affects growth factor binding and attenuates signaling through MAPK and additional pathways . Reduced signaling combined with altered E-cadherin and EGFR expression leads to reduced viability, adhesion, migration, and invasion of breast cancer cells .

Notably, the effects of HS2ST1 may be cell-type dependent, as demonstrated by the differential responses observed in various breast cancer cell lines with distinct molecular profiles .

What is the relationship between HS2ST1 and cancer stem cell phenotypes?

HS2ST1 has been shown to influence cancer stem cell (CSC) phenotypes, particularly in breast cancer:

In triple-negative MDA-MB-231 breast cancer cells, HS2ST1 overexpression significantly reduces the CD44+/CD24− phenotype (a marker of breast CSCs) from 94.05% (±0.24%) in control cells to 52.83% (±1.06%) in transfected cells . Simultaneously, the number of CD44+/CD24+ cells increases from 5.82% (±0.24%) in vector control cells to 47.07% (±1.06%) in HS2ST1-overexpressing cells .

HS2ST1 overexpression also leads to significant changes in the expression levels of CSC markers:

• Increases CD24 expression on the membrane of MDA-MB-231 cells (from 0.88±0.02 to 3.28±0.09 MFI)

• Reduces CD24 expression in MCF-7 cells (from 17.1±1.03 to 14.12±0.32 MFI)

These findings suggest that HS2ST1 modulates stemness properties in a cell-type dependent manner, with more pronounced effects in aggressive triple-negative breast cancer cells compared to hormone-receptor positive MCF-7 cells .

How does HS2ST1 affect signaling pathways in cancer cells?

HS2ST1 regulates multiple signaling pathways critical for cancer cell behavior:

• MAPK Pathway: HS2ST1-overexpressing cells show reduced MAPK signaling responses to FGF-2 stimulation . The increased viability of HS2ST1-depleted cells can be reduced to control levels by pharmacological MAPK pathway inhibition, suggesting that HS2ST1 exerts its effects at least partially through MAPK signaling .

• Growth Factor Signaling: Fluorescent ligand binding studies reveal altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells . This altered binding affects downstream signaling cascades.

• Phosphokinase Signaling: Phosphokinase array screening reveals a general decrease in signaling via multiple pathways in cells overexpressing HS2ST1 .

• Wnt Signaling: HS2ST1 overexpression alters the expression of Wnt-7a and Tcf4, suggesting effects on the Wnt signaling pathway .

• Cell Adhesion: Expression of E-cadherin is modulated by HS2ST1, affecting cell-cell adhesion properties .

The MAPK inhibitors generate a phenocopy of the HS2ST1-dependent delay in scratch wound repair, further supporting the role of this pathway in mediating HS2ST1 effects on cell migration .

What are the optimal antibody dilutions for different applications of HS2ST1 detection?

Optimal antibody dilutions vary by application and should be empirically determined for each experimental system:

It is strongly recommended that researchers titrate the antibody in each testing system to obtain optimal results, as the appropriate dilution can be sample-dependent . For western blot applications, initial testing should begin with a mid-range dilution (1:1000) and be adjusted based on signal strength and background levels . For IHC applications, starting with a 1:100 dilution is recommended, with subsequent optimization based on tissue type and fixation methods .

How can contradictory results from HS2ST1 expression studies be interpreted?

Contradictory results in HS2ST1 research can stem from several factors:

• Cell-type dependent effects: HS2ST1 shows cell line-specific effects. For example, HS2ST1 overexpression significantly reduces the CD44+/CD24− phenotype in MDA-MB-231 cells but has minimal effect in MCF-7 cells .

• Experimental context: The same molecular change may produce different phenotypes depending on the experimental assay. For instance:

  • In invasion assays, HS2ST1 overexpression inhibits Matrigel invasion

  • In migration assays, HS2ST1 affects cell motility and adhesion to fibronectin and laminin

• Expression levels: The fold-change in HS2ST1 expression may influence outcomes. Studies show that a 25-37-fold increase in HS2ST1 mRNA expression is needed to observe phenotypic changes .

• Pathway interactions: HS2ST1's effects may be modulated by the activation status of other pathways. For example, MAPK inhibitors can generate a phenocopy of HS2ST1-dependent effects, suggesting interconnected pathways .

To reconcile contradictory results, researchers should:

• Specify the exact cell line and its molecular characteristics

• Report the magnitude of HS2ST1 expression changes

• Assess multiple functional assays

• Evaluate the activation status of related signaling pathways

• Consider the possibility of context-dependent effects

What techniques are available for validating HS2ST1 antibody specificity?

To ensure experimental validity, researchers should validate HS2ST1 antibody specificity using multiple approaches:

• Western blot verification: Confirm detection of a single band at the expected molecular weight (27 kDa) . Multiple bands may indicate non-specific binding or detection of different isoforms.

• Genetic validation: Compare antibody reactivity between:

  • Wild-type cells and HS2ST1 knockout or knockdown cells

  • Control cells and HS2ST1-overexpressing cells (25-37-fold increase in mRNA should produce detectable protein changes)

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to block specific binding sites and confirm signal reduction.

• Cross-reactivity testing: Test antibody against recombinant protein arrays. High-quality HS2ST1 antibodies are tested against protein arrays of 364 human recombinant protein fragments to ensure specificity .

• Tissue validation: Confirm expected staining patterns in validated positive control tissues (human lymphoma tissue for IHC; human liver, brain, and heart tissues for WB) .

Comprehensive antibody validation should include both positive controls (tissues/cells known to express HS2ST1) and negative controls (antibody diluent without primary antibody, non-specific IgG, or tissues/cells with confirmed absence of HS2ST1) .

What future research directions are emerging for HS2ST1 antibodies in cancer research?

Emerging research directions for HS2ST1 antibodies in cancer include:

• Biomarker development: HS2ST1 has been identified as a potential predictive biomarker for osteosarcoma, suggesting applications in cancer diagnostics and prognostication .

• Therapeutic targeting: Understanding HS2ST1's role in modulating cancer cell behavior and signaling pathways opens possibilities for targeted therapeutic approaches.

• Cancer stem cell research: The demonstrated effects of HS2ST1 on cancer stem cell phenotypes warrant further investigation into its potential as a target for eliminating therapy-resistant tumor cell populations .

• Pathway-specific interventions: The connection between HS2ST1 and MAPK signaling suggests that combining HS2ST1 modulation with pathway-specific inhibitors might enhance therapeutic efficacy .

• Metastasis research: Given HS2ST1's effects on cell invasion and migration, further exploration of its role in metastatic processes could yield valuable insights for preventing cancer spread .