EPSTI1 Antibody

What is EPSTI1 and what are its key structural characteristics?

EPSTI1 is a protein encoded by the EPSTI1 gene located on chromosome 13q13.3, which was initially identified as a novel gene dramatically induced by epithelial-stromal interaction in human breast cancer. The EPSTI1 protein consists of 307 amino acids with a molecular mass of approximately 35.4 kDa . The protein's sequence includes distinct regions that facilitate its interactions with other cellular components, particularly in signaling pathways related to inflammation and immune response.

In which tissues is EPSTI1 normally expressed?

EPSTI1 expression is not restricted to the breast but is found in various normal tissues. Significant expression has been documented in the spleen, small intestine, salivary glands, testes, germinal centers of lymph nodes, and placenta . This diverse tissue distribution suggests multiple physiological roles for the protein beyond its initially discovered function in breast tissue.

What are the common research applications for EPSTI1 antibodies?

EPSTI1 antibodies are primarily used in Western blotting, ELISA, and immunohistochemistry/immunofluorescence assays . In immunohistochemistry, EPSTI1 antibodies have been used to examine expression patterns in atherosclerotic plaques versus normal arteries, often in conjunction with endothelial cell markers like CD31 . In Western blotting, these antibodies help quantify EPSTI1 expression levels in response to various stimuli such as LPS treatment .

How should researchers select an appropriate EPSTI1 antibody for their experiments?

When selecting an EPSTI1 antibody, researchers should consider the specific epitope recognized by the antibody (e.g., some target the N-terminal region like amino acids 1-100 ), the host species (to avoid cross-reactivity issues in multi-protein studies), and validated applications. Monoclonal antibodies like clone 3G7 offer high specificity but may recognize limited epitopes, while polyclonal antibodies might provide broader epitope recognition but potentially increased background. The experimental approach (Western blot, IHC, IF, etc.) should guide selection, as antibodies perform differently across applications.

What is the recommended protocol for EPSTI1 immunohistochemistry in tissue samples?

For immunohistochemistry with EPSTI1 antibodies, tissues should be sectioned to approximately 4 μm, followed by dewaxing in xylene and rehydration. Antigen retrieval should be performed using 10 mM citrate buffer for 30 minutes. Block endogenous peroxidase activity with 3% H₂O₂ for 15 minutes at room temperature, then wash three times with PBS. Block with 5% bovine serum albumin for 1 hour, followed by overnight incubation with EPSTI1 antibody (e.g., 11627-1-AP at 1:100 dilution) at 4°C . After washing, apply appropriate secondary antibodies, develop the signal, and counterstain with hematoxylin. For quantification, ImageJ software can be utilized to analyze expression levels.

How can researchers effectively perform monocyte adhesion assays to study EPSTI1's role in cell-cell interactions?

To assess EPSTI1's role in monocyte-endothelial cell adhesion:

• Suspend THP-1 cells (or other monocytic cells) in serum-free medium at 1 × 10⁶ cells/mL

• Stain THP-1 cells with DiI for 10 minutes at 37°C

• Concurrently, stain HUVECs (cultured in 12-well plates) with Hoechst 33342 for 10 minutes at 37°C

• Wash both cell types three times with PBS

• Co-incubate labeled THP-1 cells with HUVECs for 4 hours at 37°C in 5% CO₂

• Wash three times with PBS to remove non-adherent cells

• Count adherent THP-1 cells from three randomly selected microscopic fields

This assay can be performed under different conditions (e.g., with EPSTI1 knockdown or overexpression) to determine how EPSTI1 affects monocyte adhesion to endothelial cells.

What controls should be included when studying EPSTI1 in inflammatory models?

When investigating EPSTI1 in inflammatory models, particularly LPS-induced inflammation, several controls are essential:

• Untreated control groups to establish baseline EPSTI1 expression

• Dose-response controls with varying LPS concentrations (e.g., 5, 10, 20 μg/mL) to determine optimal stimulation conditions

• Time-course experiments to capture temporal changes in EPSTI1 expression

• Negative control siRNA (siNC) when performing EPSTI1 knockdown experiments

• Positive controls for NF-κB pathway activation (e.g., TNF-α treatment)

• Vehicle controls for any solvents used in treatments

Additionally, cell viability assays (e.g., CCK-8) should be performed to ensure that observed effects are not due to cytotoxicity .

What techniques are most effective for EPSTI1 knockdown studies?

For EPSTI1 knockdown studies, siRNA transfection has been effectively demonstrated in multiple cell models, including human umbilical vein endothelial cells (HUVECs) and WI-38 fibroblasts . Researchers should:

• Design or obtain validated siRNAs targeting EPSTI1 mRNA

• Include appropriate negative control siRNAs (siNC)

• Optimize transfection conditions for their specific cell type

• Confirm knockdown efficiency by both RT-qPCR and Western blotting

• Assess functional outcomes at appropriate timepoints post-transfection

• Consider potential off-target effects by using multiple siRNA sequences

In LPS-induced inflammatory models, EPSTI1 knockdown has been shown to restore cell viability, inhibit apoptosis, and reduce pro-inflammatory cytokine expression, making this approach valuable for studying EPSTI1's role in inflammation .

How does EPSTI1 contribute to atherosclerosis development?

EPSTI1 appears to play a significant role in atherosclerosis development through several mechanisms:

• Increased expression: EPSTI1 is significantly upregulated in human atherosclerotic plaques compared to normal arteries, particularly in endothelial cells

• Promotion of monocyte adhesion: EPSTI1 elevates the expression of adhesion molecules VCAM-1 and ICAM-1 in endothelial cells, enhancing monocyte adhesion to the endothelium

• Mediation of inflammatory responses: EPSTI1 enhances LPS-induced expression of adhesion molecules and subsequent monocyte-endothelial cell adhesion

• NF-κB pathway regulation: EPSTI1 is regulated by LPS in a p65-dependent manner, suggesting its involvement in inflammatory signaling cascades relevant to atherosclerosis

These findings suggest that EPSTI1 may be involved in the early stages of atherosclerosis by facilitating monocyte recruitment to the arterial wall, a critical step in atherosclerotic plaque formation.

What is the role of EPSTI1 in inflammatory responses?

EPSTI1 serves as a significant regulator of inflammatory responses through several mechanisms:

• Induction by inflammatory stimuli: EPSTI1 expression is dramatically upregulated in response to LPS stimulation in a dose-dependent manner

• Regulation of pro-inflammatory cytokines: Knockdown of EPSTI1 attenuates the expression of pro-inflammatory factors including IL-1β, IL-6, and TNF-α

• Modulation of adhesion molecules: EPSTI1 increases the expression of VCAM-1 and ICAM-1, which are critical for leukocyte recruitment during inflammation

• NF-κB pathway activation: EPSTI1 promotes inflammation by enhancing the phosphorylation of p65 and IκBα, key components of the NF-κB signaling pathway

These findings collectively indicate that EPSTI1 acts as a positive regulator of inflammatory responses and may serve as a potential therapeutic target for inflammatory diseases.

How does EPSTI1 interact with the NF-κB signaling pathway?

EPSTI1 interacts with the NF-κB signaling pathway through multiple mechanisms:

• EPSTI1 triggers NF-κB signaling and its nuclear translocation by interacting with valosin-containing protein (VCP) and inducing NF-κB inhibitor alpha (IκBα) degradation

• LPS-induced EPSTI1 expression is p65-dependent, suggesting a positive feedback loop where NF-κB activation induces EPSTI1, which further enhances NF-κB signaling

• Knockdown of EPSTI1 significantly decreases the ratios of phosphorylated p65/total p65 and phosphorylated IκBα/total IκBα, indicating reduced NF-κB pathway activation

• EPSTI1 deficiency reduces the expression of NF-κB target genes, including adhesion molecules (VCAM-1, ICAM-1) and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α)

This relationship between EPSTI1 and NF-κB signaling underscores its importance in inflammatory processes and suggests that EPSTI1 inhibition could attenuate NF-κB-mediated inflammation.

What are the challenges in interpreting EPSTI1 expression data across different tissue types?

Researchers face several challenges when interpreting EPSTI1 expression data across different tissues:

• Baseline expression variation: EPSTI1 is expressed in multiple normal tissues (spleen, small intestine, salivary glands, testes, lymph nodes, placenta) , necessitating appropriate tissue-specific controls

• Cell-type specificity: Within tissues, EPSTI1 expression may vary by cell type (e.g., higher in endothelial cells of atherosclerotic plaques) , requiring single-cell or cell-type specific analyses

• Context-dependent regulation: EPSTI1 expression responds differently to inflammatory stimuli depending on the cellular context and existing pathological conditions

• Splice variants: Potential isoforms of EPSTI1 may exist and be differentially expressed across tissues, complicating interpretation without isoform-specific detection methods

• Confounding variables: In disease states, multiple factors may influence EPSTI1 expression simultaneously, making it difficult to isolate causal relationships

Researchers should address these challenges through careful experimental design, including matched controls, cell-type specific analyses, and comprehensive characterization of EPSTI1 regulation in each tissue context.

How can researchers differentiate between direct and indirect effects of EPSTI1 in signaling cascades?

Differentiating between direct and indirect effects of EPSTI1 in signaling cascades requires multifaceted approaches:

• Temporal analysis: Monitor the kinetics of EPSTI1 induction and subsequent signaling events to establish a temporal relationship (e.g., tracking p65 phosphorylation after EPSTI1 upregulation or knockdown)

• Protein-protein interaction studies: Utilize co-immunoprecipitation, proximity ligation assays, or FRET to identify direct EPSTI1 binding partners (such as the reported interaction with VCP)

• Domain mapping: Generate truncated versions of EPSTI1 to identify which regions are essential for specific signaling effects

• Rescue experiments: In EPSTI1 knockdown models, reintroduce wild-type or mutant EPSTI1 to determine which domains are necessary for restoring signaling activities

• Parallel pathway analysis: Simultaneously monitor multiple signaling pathways to identify specific versus global effects of EPSTI1 modulation

• Transcriptome and proteome profiling: Compare immediate versus delayed gene/protein expression changes after EPSTI1 modulation to distinguish primary from secondary effects

These approaches collectively can help establish whether EPSTI1 directly participates in signaling cascades or acts indirectly through intermediate molecules.

What approaches can help resolve contradictory findings regarding EPSTI1 function?

Resolving contradictory findings about EPSTI1 function requires systematic investigation:

• Cell type considerations: Determine if contradictions arise from cell-type specific functions of EPSTI1, as its roles may differ between epithelial cells, endothelial cells, and immune cells

• Context-dependent analysis: Examine whether EPSTI1 functions differently under various physiological or pathological conditions (e.g., inflammatory versus homeostatic states)

• Dose-dependent effects: Assess whether EPSTI1 exhibits different or even opposing functions at different expression levels

• Temporal dynamics: Investigate if EPSTI1's function changes over time during disease progression or cellular responses

• Isoform-specific studies: Determine whether different EPSTI1 isoforms or post-translational modifications account for contradictory findings

• Methodological harmonization: Compare experimental methods across studies and adopt standardized protocols for EPSTI1 manipulation and assessment

• Integrative analysis: Combine data from multiple experimental models (in vitro, ex vivo, and in vivo) to develop a unified understanding of EPSTI1 biology

Through these approaches, researchers can develop a more nuanced understanding of EPSTI1's multifaceted roles in different biological contexts.

How should researchers address non-specific binding when using EPSTI1 antibodies?

To minimize non-specific binding with EPSTI1 antibodies:

• Antibody validation: Verify antibody specificity using positive controls (tissues/cells known to express EPSTI1) and negative controls (EPSTI1 knockdown samples)

• Blocking optimization: Test different blocking solutions (5% BSA, 10% goat serum, etc.) and durations (1-2 hours) to reduce background

• Antibody titration: Perform dilution series experiments to determine optimal antibody concentration that maximizes specific signal while minimizing background

• Washing protocol refinement: Increase washing frequency or duration with PBS after antibody incubation

• Secondary antibody controls: Include controls omitting primary antibody to identify potential secondary antibody non-specific binding

• Pre-absorption: Consider pre-absorbing the antibody with recombinant EPSTI1 protein to confirm specificity

• Alternative antibody selection: If possible, test antibodies from different sources or those targeting different epitopes of EPSTI1

When performing double immunostaining, as with EPSTI1 and CD31, additional controls should be included to ensure that signals are not the result of cross-reactivity between detection systems .

What factors might contribute to variability in EPSTI1 expression across experimental replicates?

Several factors can contribute to variability in EPSTI1 expression measurements:

Researchers should standardize experimental conditions, include appropriate controls in each experiment, and consider pooling data from multiple independent experiments to address these sources of variability.

How can researchers optimize EPSTI1 detection in cells with low baseline expression?

For detecting EPSTI1 in cells with low baseline expression:

• Signal amplification: Consider using signal amplification techniques such as tyramide signal amplification for immunostaining or highly sensitive chemiluminescent substrates for Western blotting

• Protein concentration: Increase the amount of total protein loaded for Western blotting or use protein concentration methods prior to analysis

• Pre-induction: If appropriate for the experimental question, consider pre-treating cells with known EPSTI1 inducers like LPS or IFN-γ before analysis

• Enhanced imaging: Use confocal microscopy with increased exposure times or gain settings while maintaining controls to account for background

• Enrichment strategies: Consider subcellular fractionation to concentrate EPSTI1 from its predominant cellular compartment

• RT-qPCR optimization: For mRNA detection, optimize primer design and use highly sensitive reverse transcription kits

• Alternative detection methods: Consider using more sensitive techniques like droplet digital PCR or mass spectrometry-based proteomics

When using these approaches, appropriate controls must be included to distinguish between true low-level expression and technical artifacts.