ELF4 Antibody

What is ELF4 and why is it important in research?

ELF4, also known as myeloid Elf-1-like factor (MEF), is a 663 amino acid member of the Ets-1 family of transcription factors characterized by a conserved DNA binding domain. It plays crucial roles in regulating cellular processes such as proliferation, differentiation, and angiogenesis . ELF4 is particularly significant in research due to its involvement in activating promoters of hematopoietic growth factor genes, including GM-CSF, IL-3, and IL-8, as well as its implication in the activation of the Perforin 1 promoter in natural killer (NK) cells . Recent studies have also highlighted its role in alleviating inflammatory bowel disease, making it a promising target for therapeutic interventions .

What applications are ELF4 antibodies suitable for?

ELF4 antibodies, such as the F-11 mouse monoclonal antibody, are versatile tools suitable for multiple experimental applications including:

• Western blotting (WB): For detecting ELF4 protein expression levels in tissue or cell lysates

• Immunoprecipitation (IP): For isolating ELF4 protein complexes

• Immunofluorescence (IF): For visualizing ELF4 cellular localization

• Enzyme-linked immunosorbent assay (ELISA): For quantitative detection of ELF4

These applications make ELF4 antibodies essential for researchers studying its expression patterns, protein interactions, and functional roles in various biological contexts.

In which species and tissues is ELF4 antibody reactivity confirmed?

The F-11 ELF4 antibody demonstrates confirmed reactivity across multiple species including mouse, rat, and human samples . Regarding tissue distribution, ELF4 is predominantly localized in the nucleus and shows high expression in the placenta and myeloid leukemia cells. Lower expression levels have been documented in tissues such as the lung, heart, thymus, spleen, colon, ovary, and peripheral blood lymphocytes . This cross-species reactivity makes it particularly valuable for comparative studies and translational research.

How can I optimize ChIP experiments using ELF4 antibody?

For optimal Chromatin Immunoprecipitation (ChIP) experiments using ELF4 antibody, follow these methodological steps based on validated protocols:

• Cross-linking: Treat cells with 1% formaldehyde for 10 minutes and terminate the reaction with glycine solution

• Cell collection: Harvest cells in PBS containing protease inhibitors

• Sonication: Fragment chromosomes into 200-1000 bp segments using 15 cycles of 10-second bursts with 10-second intervals

• Immunoprecipitation: After centrifugation (12,000g, 4°C, 10 minutes), divide the supernatant and incubate overnight at 4°C with:

  • ELF4-specific antibody (1μg, sc-390689)

  • Negative control IgG (ab205718)

• Washing: Remove nonspecific complexes through centrifugation and washing

• Reverse cross-linking: Incubate overnight at 65°C

• DNA purification: Extract DNA fragments using phenol/chloroform

• Analysis: Detect ELF4 enrichment on target promoters using qPCR

This protocol has been successfully employed to demonstrate ELF4 binding to the IL1RN promoter, confirming its transcriptional regulatory function.

What controls should be included when studying ELF4-mediated transcriptional regulation?

When investigating ELF4-mediated transcriptional regulation, incorporate these essential controls:

• Negative antibody control: Always include an IgG control (e.g., ab205718) for ChIP experiments to account for non-specific binding

• Promoter binding site mutants: Generate mutated versions of the predicted ELF4 binding sites in promoter-reporter constructs. For example, when studying IL1RN regulation, researchers successfully created pGL3-IL1RN Wild Type (WT) and pGL3-IL1RN Mutant (MUT) constructs

• Gene expression manipulation controls:

  • Silencing controls (sh-NC vs. sh-ELF4)

  • Overexpression controls (oe-NC vs. oe-ELF4)

• Dual-luciferase reporter system: Include both experimental (firefly luciferase) and internal control (Renilla luciferase) reporters to normalize for transfection efficiency. Calculate relative luciferase activity as the ratio of firefly to Renilla luciferase activity (FL/RL)

These controls ensure reliable interpretation of ELF4's transcriptional regulatory functions.

How can I validate the specificity of ELF4 antibody for my particular application?

To validate ELF4 antibody specificity for your specific application, implement this comprehensive approach:

• Positive and negative tissue/cell controls:

  • Use tissues/cells known to express high levels of ELF4 (placenta, myeloid leukemia cells) as positive controls

  • Use tissues with lower expression (e.g., lung, heart) as comparative references

• Antibody concentration titration:

  • Perform dilution series to determine optimal antibody concentration for your specific application

  • Standard starting concentrations: 200 μg/ml for western blotting, adjusting as needed

• Overexpression and knockdown validation:

  • Compare antibody reactivity in cells with:

    • ELF4 overexpression (using oe-ELF4 plasmids)

    • ELF4 knockdown (using sh-ELF4 constructs)

    • Appropriate negative controls (oe-NC, sh-NC)

• Western blot analysis:

  • Confirm a single band of appropriate molecular weight (663 amino acids)

  • Use GAPDH as loading control

  • Calculate relative expression as the ratio of target band intensity to internal reference band

This systematic validation ensures antibody performance is optimized for your specific experimental conditions.

How does ELF4 regulate gene expression through promoter binding?

ELF4 regulates gene expression through a defined mechanism of promoter binding and transcriptional activation:

• Sequence-specific DNA binding: ELF4, as an ETS family transcription factor, recognizes and binds to specific DNA sequences in target gene promoters. For example, the JASPAR database has been used to predict ELF4 binding sites on the IL1RN promoter .

• Transcriptional activation: Once bound to promoter regions, ELF4 enhances transcription of target genes. This has been experimentally validated using dual-luciferase reporter assays, where:

  • Silencing ELF4 significantly reduced IL1RN promoter activity

  • Overexpressing ELF4 significantly increased IL1RN promoter activity

  • Mutating the ELF4 binding site eliminated these effects

• Confirmation through chromatin immunoprecipitation: ChIP experiments have demonstrated that:

  • Silencing ELF4 decreased enrichment of ELF4 at the IL1RN promoter

  • Overexpressing ELF4 significantly increased enrichment at the IL1RN promoter

These findings collectively establish that ELF4 functions as a direct transcriptional activator of target genes through sequence-specific promoter binding.

What is the role of ELF4 in inflammatory processes and macrophage polarization?

ELF4 plays a significant role in modulating inflammatory processes, particularly through its effects on macrophage polarization:

• Regulation of anti-inflammatory mediators: ELF4 directly enhances transcription of IL1RN (IL-1 receptor antagonist), an important anti-inflammatory cytokine. Experimental evidence shows a positive correlation between ELF4 and IL1RN expression levels .

• Macrophage polarization control: ELF4 influences the balance between pro-inflammatory M1 and anti-inflammatory M2 macrophage phenotypes:

  • Overexpression of ELF4 increases the proportion of M2 macrophages while decreasing M1 macrophages

  • This effect is mediated through IL1RN, as silencing IL1RN reverses the macrophage polarization shift induced by ELF4 overexpression

• Molecular markers: The ELF4/IL1RN axis affects macrophage polarization markers:

  • M2 markers (Arg1, CD163): Elevated by ELF4 overexpression, reduced when IL1RN is silenced

  • M1 marker (iNOS): Decreased by ELF4 overexpression, increased when IL1RN is silenced

These findings suggest ELF4 could be a therapeutic target for inflammatory disorders, particularly inflammatory bowel disease, through its promotion of anti-inflammatory macrophage polarization.

How can I investigate ELF4's role in inflammatory bowel disease models?

To investigate ELF4's role in inflammatory bowel disease (IBD) models, implement this comprehensive experimental approach:

• In vivo model establishment:

  • Utilize LPS-induced IBD mouse models

  • Create experimental groups: Control, LPS treatment, LPS+oe-NC (overexpression negative control), and LPS+oe-ELF4 (ELF4 overexpression)

• Gene expression manipulation:

  • Design and synthesize ELF4-specific shRNA for knockdown studies

  • Create oe-ELF4 plasmids for overexpression studies

  • Include appropriate controls (sh-NC, oe-NC)

• Tissue and cellular analysis:

  • Analyze colon tissue for ELF4 and downstream target (e.g., IL1RN) expression using:

    • RT-qPCR for mRNA levels

    • Western blot for protein levels

  • Isolate and analyze bone marrow-derived macrophages (BMDMs) to study:

    • Macrophage polarization by flow cytometry (CD68+CD80+ for M1, CD68+CD206+ for M2)

    • Expression of polarization markers (iNOS for M1; Arg1, CD163 for M2)

• Molecular mechanism investigation:

  • Perform ChIP to determine ELF4 binding to target gene promoters

  • Use dual-luciferase reporter assays with wild-type and mutant promoter constructs

  • Establish rescue experiments (e.g., oe-ELF4 + sh-IL1RN) to confirm downstream mediators

• Inflammatory readouts:

  • Measure inflammatory cytokines (Treml, IL-6, TNF-α, IL-1β) by ELISA

  • Assess epithelial cell apoptosis by flow cytometry and apoptotic markers (Bcl-2, Bax, caspases)

This multifaceted approach enables comprehensive characterization of ELF4's role in IBD pathophysiology.

Why might I observe differential ELF4 expression across tissue samples?

Differential ELF4 expression across tissue samples can result from multiple factors that require careful experimental interpretation:

• Normal biological variation: ELF4 naturally exhibits tissue-specific expression patterns, with higher levels in placenta and myeloid leukemia cells, and lower levels in tissues such as lung, heart, thymus, spleen, colon, ovary, and peripheral blood lymphocytes . This biological variation reflects tissue-specific transcriptional programs.

• Pathological conditions: Expression may be altered in disease states, particularly inflammatory conditions or malignancies where transcription factor networks are dysregulated. For instance, ELF4 levels may change in inflammatory bowel disease contexts .

• Experimental factors:

  • Sample preparation variations: Inconsistent fixation, extraction, or preservation methods

  • Antibody specificity: The F-11 ELF4 antibody (sc-390689) recognizes specific epitopes that may be differentially accessible in various tissue contexts

  • Detection sensitivity: Different applications (WB, IF, IP, ELISA) have varying detection thresholds

• Verification approaches:

  • Multi-antibody validation: Use alternative ELF4 antibodies targeting different epitopes

  • Transcript-protein correlation: Compare protein detection (Western blot) with mRNA levels (RT-qPCR)

  • Loading controls: Normalize to appropriate housekeeping proteins (e.g., GAPDH)

Understanding these factors ensures accurate interpretation of ELF4 expression patterns across different experimental contexts.

How should I analyze contradictory results between ELF4 antibody-based detection methods?

When facing contradictory results between different ELF4 antibody-based detection methods, employ this systematic troubleshooting approach:

• Method-specific considerations:

  Detection Method	Potential Issues	Validation Approach
  Western Blot	Denaturation may affect epitope recognition	Use reducing vs. non-reducing conditions
  Immunofluorescence	Fixation can mask epitopes	Compare different fixation methods (PFA vs. methanol)
  ChIP	Crosslinking efficiency varies	Optimize formaldehyde concentration and time
  ELISA	Tertiary protein structure requirements	Test different antibody pairs

• Antibody characteristics assessment:

  • Determine if the ELF4 antibody (e.g., F-11, sc-390689) recognizes native or denatured protein

  • Review epitope location (N-terminal, C-terminal, internal)

  • Check for potential cross-reactivity with related ETS family members

• Sample preparation harmonization:

  • Standardize protein extraction protocols

  • Use the same samples across different detection methods

  • Perform parallel positive and negative controls

• Quantitative comparison:

  • Normalize results using appropriate controls for each method

  • Calculate relative expression rather than absolute values

  • Apply statistical analysis to determine significance of differences

This systematic approach helps reconcile contradictory results and identify the most reliable detection method for your specific research question.

What are the critical parameters for optimizing Western blot detection of ELF4?

For optimal Western blot detection of ELF4, pay careful attention to these critical parameters:

• Gel percentage selection:

  • Use 8-12% SDS gels based on ELF4's size (663 amino acids)

  • Lower percentage gels provide better resolution for this relatively large protein

• Protein extraction optimization:

  • For nuclear proteins like ELF4, use specialized nuclear extraction protocols

  • Include protease inhibitors to prevent degradation

  • Maintain cold conditions throughout extraction

• Antibody concentration and incubation:

  • Start with recommended concentration (200 μg/ml) for primary antibody

  • Incubate primary antibody (ELF4, sc-390689) overnight at 4°C

  • Use appropriate secondary antibody (goat anti-mouse IgG-HRP, 1:5000)

  • Include sufficient washing steps (3× with TBST, 5 minutes each)

• Blocking conditions:

  • Use 5% skim milk or 5% BSA for blocking

  • Block for 1 hour at room temperature to minimize background

• Detection and quantification:

  • Use ECL reagent with appropriate exposure time (starting with 1 minute)

  • Capture images using a gel imaging system (e.g., Image Quant LAS 4000C)

  • Normalize to GAPDH or other appropriate housekeeping protein

  • Calculate relative expression as the ratio of target to reference band intensity

These optimized parameters enable consistent and sensitive detection of ELF4 protein across experimental conditions.

How can I investigate ELF4's interaction with other transcription factors?

To investigate ELF4's interactions with other transcription factors, implement these advanced experimental approaches:

• Co-immunoprecipitation (Co-IP):

  • Use ELF4 antibody (sc-390689) to pull down ELF4 complexes

  • Analyze precipitated proteins by Western blot using antibodies against suspected interacting partners

  • Include appropriate controls: IgG negative control, input samples, and reciprocal Co-IP

• Proximity ligation assay (PLA):

  • Visualize in situ protein-protein interactions at single-molecule resolution

  • Use primary antibodies from different species against ELF4 and potential partners

  • Quantify interaction signals in different cellular compartments

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Perform parallel ChIP-seq for ELF4 and other transcription factors

  • Identify genomic regions with overlapping binding patterns

  • Analyze motif co-occurrence in shared regulatory regions

  • Follow the validated ChIP protocol using ELF4 antibody (sc-390689)

• Sequential ChIP (Re-ChIP):

  • First ChIP with ELF4 antibody

  • Second ChIP on the same material with antibody against potential partner

  • Only regions bound by both factors will be enriched

  • Use qPCR primers targeting specific promoters (e.g., IL1RN)

These complementary approaches can reveal both physical interactions and functional cooperation between ELF4 and other transcription factors in regulating target gene expression.

How can I evaluate the role of ELF4 in macrophage-epithelial cell interactions?

To evaluate ELF4's role in macrophage-epithelial cell interactions, employ this comprehensive co-culture experimental framework:

• Co-culture system establishment:

  • Prepare bone marrow-derived macrophages (BMDMs) with modified ELF4 expression:

    • Control: oe-NC + sh-NC

    • ELF4 overexpression: oe-ELF4 + sh-NC

    • ELF4/IL1RN pathway modulation: oe-ELF4 + sh-IL1RN

  • Culture with intestinal epithelial cells (e.g., MODE-K cell line)

• Direct interaction studies:

  • Use transwell co-culture systems to distinguish contact-dependent from secreted factor effects

  • Analyze epithelial cell parameters:

    • Apoptosis by flow cytometry

    • Expression of apoptosis regulators (Bcl-2, Bax, caspases, Cytochrome c) by Western blot

• Conditioned media experiments:

  • Collect supernatants from macrophages with modified ELF4 expression

  • Apply to epithelial cells and assess responses

  • Measure inflammatory mediators (Treml, IL-6, TNF-α, IL-1β) by ELISA

• Molecular pathway analysis:

  • Determine if the ELF4/IL1RN axis in macrophages affects epithelial cell function

  • Assess whether silencing IL1RN negates the protective effects of ELF4 overexpression

  • Analyze epithelial barrier proteins and tight junction components

• In vivo validation:

  • Create mouse models with macrophage-specific ELF4 overexpression or knockdown

  • Analyze intestinal inflammation, barrier function, and epithelial integrity

  • Perform histological analysis of macrophage-epithelial interactions

This approach provides mechanistic insights into how ELF4-mediated macrophage polarization influences epithelial cell function in inflammatory contexts.

What are the emerging applications of ELF4 antibodies in cancer research?

ELF4 antibodies are becoming increasingly valuable tools in cancer research, with several emerging applications:

• Diagnostic and prognostic biomarker development:

  • ELF4 is highly expressed in myeloid leukemia cells, suggesting potential utility as a diagnostic marker

  • Immunohistochemistry using ELF4 antibodies can help stratify patients based on expression levels

  • Correlation of expression patterns with clinical outcomes may identify prognostic signatures

• Therapeutic target validation:

  • ELF4's role in activating promoters of hematopoietic growth factor genes (GM-CSF, IL-3, IL-8) suggests involvement in cancer progression

  • Its implication in the activation of Perforin 1 promoter in NK cells indicates potential immunoregulatory functions relevant to cancer immunotherapy

  • ELF4 antibodies can be used to validate inhibition strategies in preclinical models

• Tumor microenvironment characterization:

  • Given ELF4's influence on macrophage polarization, antibodies can help assess tumor-associated macrophage phenotypes

  • Multiplex immunofluorescence with markers of M1/M2 polarization (iNOS, Arg1, CD163) and ELF4 can map spatial relationships in tumor tissues

  • This may reveal mechanisms of immune evasion or immunotherapy resistance

• Functional genomics screening:

  • ELF4 antibodies can validate CRISPR or RNAi screens targeting pathways involved in tumorigenesis

  • ChIP-seq applications using ELF4 antibodies can identify cancer-specific regulatory networks

  • Analysis of ELF4 binding to oncogene promoters may reveal novel therapeutic targets

These emerging applications position ELF4 antibodies as valuable tools in understanding cancer biology and developing new therapeutic approaches.