PEBP4 Antibody

What is PEBP4 and why is it significant for immunological research?

PEBP4 is a cytoplasmic alkaline protein with multiple biological functions and high expression in mammals. It belongs to the phosphatidylethanolamine binding protein family and has been identified as both an intracellular protein and a secreted protein . The significance of PEBP4 in research stems from its involvement in several critical biological processes:

• Anti-inflammatory and hepatoprotective effects in liver injury and fibrosis models

• Regulation of apoptosis in various cell types

• Modulation of signaling pathways including PI3K/AKT, MAPK/ERK, and NF-κB

• Association with various cancers including hepatocellular carcinoma and non-small cell lung cancer

• Recently identified role in acute lung injury models and alveolar fluid clearance

Understanding PEBP4's functions provides insights into disease mechanisms and potential therapeutic targets, making PEBP4 antibodies essential research tools.

What are the recommended applications for PEBP4 antibodies?

Based on validated research protocols, PEBP4 antibodies are suitable for multiple applications:

• Western blotting (1-3 μg/ml concentration) for detecting approximately 26 kDa PEBP4 protein

• Immunohistochemistry (5-10 μg/ml) for both frozen and paraffin-embedded tissues

• Immunohistochemistry-paraffin (5-10 μg/ml) with successful detection in human pancreas tissue

• Peptide ELISA with detection limit of 1:4000 dilution

• Co-immunoprecipitation assays for studying protein-protein interactions, particularly with signaling molecules like Akt, mTORC1, and mTORC2

When selecting applications, researchers should consider that an additional band of unknown identity at approximately 30 kDa is consistently observed in western blots using some PEBP4 antibodies. This band can be successfully blocked by incubation with the immunizing peptide, suggesting potential isoforms or post-translational modifications .

How should researchers validate PEBP4 antibody specificity?

Proper validation of PEBP4 antibody specificity is critical for reliable research outcomes. Recommended validation steps include:

• Peptide competition assays: Incubating the antibody with its immunizing peptide (e.g., peptide with sequence C-RERASEPKHKNQAE corresponding to C-terminus according to NP_659399.2) should block specific binding .

• Positive and negative tissue controls: Human heart lysates have shown consistent PEBP4 expression and should be used as positive controls. Human pancreas shows staining consistent with lysosomal localization .

• Knockout/knockdown validation: Compare antibody reactivity in wild-type versus PEBP4 knockout or knockdown samples. Studies using PEBP4 knockout mice or cell lines with PEBP4 knockdown provide excellent specificity controls .

• Multiple antibody comparison: When possible, compare results from different PEBP4 antibodies targeting distinct epitopes.

• Cross-reactivity assessment: While some PEBP4 antibodies are human-specific, researchers working with animal models should verify cross-reactivity, noting that mouse PEBP4 shows only 44.6% sequence identity with human PEBP4 .

How can researchers use PEBP4 antibodies to investigate signaling pathway interactions?

PEBP4 interacts with multiple signaling pathways, particularly PI3K/AKT, MAPK/ERK, and mTOR. To effectively study these interactions:

• Co-immunoprecipitation (Co-IP): Use PEBP4 antibodies for Co-IP followed by immunoblotting for pathway components. Research has demonstrated physical association of PEBP4 with Akt, mTORC1, and mTORC2 using this approach .

• Pathway inhibitor studies: Combine PEBP4 antibody detection with specific pathway inhibitors (e.g., MK2206 for Akt inhibition) to delineate hierarchical relationships. For example, MK2206 has been shown to suppress PEBP4 overexpression effects on mTORC1 but not mTORC2 activity .

• Phosphorylation-specific detection: Use phospho-specific antibodies for pathway components (p-Akt, p-ERK, p-IκB-α) alongside PEBP4 detection to correlate PEBP4 levels with pathway activation states. Studies show PEBP4 deficiency can increase the p-IκB-α/IκB-α ratio and p-NF-κB p65/NF-κB p65 ratio .

• Rescue experiments: Utilize constitutively active mutants (e.g., AktS473D) in PEBP4-manipulated systems to determine pathway dependencies. Research has shown that introduction of AktS473D mutant can rescue mTORC1 activity in PEBP4-knockdown cells .

These approaches can help resolve contradictory findings about PEBP4's role in different cellular contexts, such as its reported pro-survival effects in cancer cells versus protective effects in liver and lung injury models.

What methodological considerations are important when using PEBP4 antibodies in disease models?

When investigating PEBP4's role in disease models, researchers should consider:

For acute lung injury (ALI) models:

• Combine immunohistochemistry with bronchoalveolar lavage fluid (BALF) analysis to correlate PEBP4 expression with disease progression

• Include detection of AFC-related markers (ENaC-α, ENaC-γ, Na,K-ATPase α1, Na,K-ATPase β1) alongside PEBP4 to investigate functional relationships

• Consider dual staining to determine PEBP4 localization in specific lung cell populations, particularly type II alveolar epithelial cells where PEBP4 has been identified as a marker

For liver fibrosis models:

• Correlate PEBP4 antibody staining with fibrosis markers (α-SMA, collagen I and collagen III) in CCl4-induced liver fibrosis models

• Use matched tissue sections for PEBP4 immunohistochemistry and Masson staining to associate PEBP4 expression with collagen deposition

• Include biochemical measurements (ALT, AST, HYP activities) alongside PEBP4 detection to correlate protein expression with functional outcomes

For cancer research:

• Differentiate between tissue-specific effects, as PEBP4 functions may vary between cancer types

• Consider cell migration/invasion assays in conjunction with PEBP4 immunolabeling to correlate expression with metastatic potential

• Include epithelial-mesenchymal transition (EMT) markers alongside PEBP4 detection, as PEBP4 has been linked to EMT regulation through Akt pathways

How can researchers address contradictory findings regarding PEBP4 function in different models?

PEBP4 shows seemingly contradictory functions across different studies - protective in liver and lung injury models but potentially promoting cancer progression. To address these contradictions:

• Context-dependent signaling analysis: Simultaneously analyze multiple pathways (PI3K/AKT, MAPK/ERK, NF-κB) in different models to determine if PEBP4 preferentially activates different pathways in different contexts .

• Temporal expression profiling: Use PEBP4 antibodies for time-course studies to determine if PEBP4's function changes during disease progression. For example, in CCl4-induced liver fibrosis, PEBP4 expression decreases over time, suggesting dynamic regulation .

• Isoform-specific detection: Consider whether different tissues may express different PEBP4 isoforms. The consistent observation of a 30 kDa band in addition to the expected 26 kDa band in western blots suggests potential isoforms .

• Subcellular localization studies: Combine fractionation techniques with PEBP4 antibody detection to determine if PEBP4's function correlates with its subcellular localization. PEBP4 has been reported as both a cytoplasmic and secreted protein, potentially with different functions .

• Genetic manipulation confirmation: Use both overexpression and knockdown/knockout approaches to confirm findings. Studies have shown that PEBP4 deficiency exacerbates liver fibrosis and acute lung injury, while overexpression provides protection .

What are the optimal conditions for Western blotting with PEBP4 antibodies?

For optimal Western blotting results with PEBP4 antibodies:

• Sample preparation: Use RIPA buffer for protein extraction, as demonstrated in successful detection of PEBP4 in human heart lysates .

• Protein loading: Load 35 μg protein per lane for adequate detection of the approximately 26 kDa PEBP4 band .

• Antibody concentration: Use 1-3 μg/ml of PEBP4 antibody for Western blotting .

• Incubation conditions: Primary antibody incubation for 1 hour has been successful in published protocols .

• Detection method: Chemiluminescence detection provides sufficient sensitivity for PEBP4 visualization .

• Anticipated results: Expect a primary band at approximately 26 kDa (calculated MW of 25.7 kDa according to NP_659399.2). Be aware that an additional band of unknown identity at approximately 30 kDa is often observed. This band can be blocked by incubation with the immunizing peptide, suggesting it may represent a PEBP4 variant or post-translational modification .

• Controls: Include both positive controls (human heart lysate) and negative controls (primary antibody omission and/or peptide competition) .

What are the methodological considerations for immunohistochemistry with PEBP4 antibodies?

For successful immunohistochemistry using PEBP4 antibodies:

• Antibody concentration: Use 5-10 μg/ml for both frozen and paraffin-embedded tissues .

• Antigen retrieval: For paraffin sections, heat-induced epitope retrieval is recommended given the cytoplasmic localization of PEBP4.

• Tissue selection: Human pancreas has shown reliable staining and can serve as a positive control tissue. The staining pattern is consistent with lysosomal localization .

• Signal amplification: Consider using biotin-streptavidin amplification systems for enhanced sensitivity, especially when detecting low PEBP4 expression levels.

• Counter-staining: Hematoxylin counterstaining provides good nuclear contrast to cytoplasmic PEBP4 staining.

• Anticipated results: In human pancreas, expect staining consistent with lysosomal localization . In liver sections, PEBP4 shows cytoplasmic staining in hepatocytes .

• Controls: Include peptide competition controls and PEBP4 knockout tissues when available to confirm specificity.

How can researchers overcome common challenges in PEBP4 detection?

Researchers frequently encounter challenges when working with PEBP4 antibodies. Here are strategies to address common issues:

• Low signal intensity:

  • Increase antibody concentration within the recommended range (1-3 μg/ml for WB; 5-10 μg/ml for IHC)

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

  • Use signal amplification methods (HRP-polymer detection systems)

  • Increase protein loading (up to 50 μg per lane for WB)

• Background issues:

  • Include longer blocking steps (5% non-fat dry milk or BSA for 2 hours)

  • Add 0.1-0.5% Triton X-100 to reduce non-specific binding

  • Perform peptide competition controls to distinguish specific from non-specific signals

  • Use monoclonal antibodies if polyclonal antibodies show high background

• Multiple bands/non-specific binding:

  • The additional 30 kDa band often observed with PEBP4 antibodies can be blocked with immunizing peptide

  • Increase washing duration and volume between antibody incubations

  • Optimize antibody dilution through titration experiments

  • Consider using freshly prepared samples to minimize degradation products

• Tissue-specific optimization:

  • For lung tissue, perfusion fixation produces better results than immersion fixation

  • For liver sections, shorter fixation times (4-6 hours) may preserve PEBP4 antigenicity better

  • For cancer tissues, dual staining with proliferation markers can help contextualize PEBP4 expression

How can PEBP4 antibodies be utilized to investigate acute lung injury mechanisms?

PEBP4 antibodies provide valuable insights into acute lung injury (ALI) pathophysiology:

• Correlation with disease severity: Immunohistochemistry with PEBP4 antibodies can assess PEBP4 expression in relation to pathological damage in LPS-induced ALI mouse models. Research has shown that PEBP4 deficiency exacerbates lung pathological damage and edema .

• Signaling pathway investigation: Western blotting with PEBP4 antibodies alongside phospho-specific antibodies for PI3K/AKT pathway components can reveal how PEBP4 influences this pathway in ALI. Studies indicate that PEBP4 deletion enhances suppression of the PI3K/AKT signaling pathway in ALI models .

• Cell-specific expression patterns: Immunofluorescence co-labeling with PEBP4 antibodies and cell-type-specific markers can identify which pulmonary cell populations express PEBP4. PEBP4 has been identified as a marker of type II alveolar epithelial cells (AECs), whose dysfunction is involved in ALI progression .

• Alveolar fluid clearance (AFC) mechanism study: Co-immunoprecipitation with PEBP4 antibodies can reveal interactions with AFC-related proteins like ENaC-α, ENaC-γ, Na,K-ATPase α1, and Na,K-ATPase β1. Research shows PEBP4 deletion reduces expression of these AFC-related markers in ALI models .

• Intervention studies: PEBP4 antibodies can monitor protein expression changes in response to therapeutic interventions. For example, selective PI3K/AKT pathway activators (740YP or SC79) partially rectify the effects of PEBP4 deletion in ALI models .

What approaches can researchers use to study PEBP4's role in cancer progression?

PEBP4 has demonstrated complex roles in cancer progression that can be investigated through several antibody-based approaches:

• Expression correlation with malignancy: Immunohistochemistry with PEBP4 antibodies can assess expression patterns across cancer stages. Studies indicate PEBP4 expression increases in many cancer specimens and correlates with cancer progression .

• Mechanistic pathway analysis: Western blotting with PEBP4 antibodies alongside key signaling molecules reveals pathway relationships. Research shows knockdown of PEBP4 in hepatocellular carcinoma cells diminishes activities of Akt, mTORC1, and mTORC2 .

• Protein interaction profiling: Co-immunoprecipitation using PEBP4 antibodies followed by mass spectrometry can identify novel interaction partners. Physical association of PEBP4 with Akt, mTORC1, and mTORC2 has been observed using this approach .

• Functional phenotype correlation: Combining PEBP4 antibody detection with cell proliferation, migration, and invasion assays links expression to functional outcomes. Research demonstrates that PEBP4 knockdown-engendered reduction of cell proliferation, migration, and invasion can be partially rescued by constitutively active Akt .

• In vivo tumor model analysis: Immunohistochemistry with PEBP4 antibodies in xenograft models correlates expression with tumor growth and metastasis. Knockdown of PEBP4 has been shown to diminish tumor growth and metastasis, whereas overexpression enhances these parameters .

• EMT marker correlation: Multiplex immunofluorescence with PEBP4 and EMT markers can reveal relationships with cancer cell plasticity. The expression of EMT markers appears to be fully regulated by Akt in PEBP4-overexpressing cells .

How can researchers investigate PEBP4's role in liver fibrosis using antibody-based techniques?

PEBP4 antibodies offer multiple approaches to study its role in liver fibrosis:

• Expression profiling during fibrosis progression: Western blotting has demonstrated that PEBP4 is downregulated in the liver tissue of CCl4-treated wild-type mice compared with control mice, suggesting dynamic regulation during fibrosis development .

• Cellular localization in fibrotic liver: Immunohistochemistry can identify PEBP4-expressing cell populations in fibrotic liver tissue. Hepatocyte-conditional knockout mice (PEBP4 flox/flox, Alb-Cre mice) have been used to study hepatocyte-specific PEBP4 functions .

• Correlation with fibrosis markers: Co-immunostaining or sequential section staining with PEBP4 antibodies and fibrosis markers (α-SMA, collagen I, collagen III) can correlate expression patterns. PEBP4 deficiency has been shown to enhance the upregulation of these ECM markers in CCl4-treated mice .

• NF-κB pathway activation assessment: Western blotting for PEBP4 alongside NF-κB pathway components can reveal regulatory relationships. PEBP4 knockout results in increased expression of nuclear NF-κB p65, elevated p-IκB-α/IκB-α ratio, and higher p-NF-κB p65/NF-κB p65 ratio in liver fibrosis models .

• Intervention studies: PEBP4 antibodies can monitor expression changes in response to anti-fibrotic interventions, potentially identifying new therapeutic approaches for liver fibrosis.

What are the future prospects for PEBP4 antibody applications in research?

Emerging research directions for PEBP4 antibody applications include:

• Single-cell analysis: Integration of PEBP4 antibodies with single-cell technologies (CyTOF, imaging mass cytometry) could reveal cell-specific expression patterns and heterogeneity within tissues.

• Secreted versus intracellular PEBP4: Development of antibodies specifically distinguishing between secreted and intracellular forms of PEBP4 would clarify its dual functions. Several reports indicate PEBP4 functions as both a secreted and cytoplasmic protein .

• Post-translational modification mapping: Phospho-specific PEBP4 antibodies could reveal how PEBP4's function is regulated by post-translational modifications.

• In vivo imaging: Development of fluorescently-labeled PEBP4 antibodies for intravital microscopy could enable real-time tracking of PEBP4 expression during disease progression.

• Therapeutic targeting: PEBP4 antibodies could potentially neutralize secreted PEBP4 in cancers where it promotes progression, while delivery systems could increase PEBP4 levels in contexts where it provides protection (e.g., liver injury, acute lung injury).

• Multi-omics integration: Combining PEBP4 antibody-based proteomics with transcriptomics and metabolomics could provide comprehensive understanding of PEBP4's role in cellular homeostasis.

• Cross-species comparative studies: Development of antibodies recognizing conserved PEBP4 epitopes across species would facilitate translational research, particularly important given the relatively low (44.6%) sequence identity between mouse and human PEBP4 .

How can researchers integrate PEBP4 antibody data with other -omics approaches?

Integrating PEBP4 antibody data with other -omics approaches can provide comprehensive insights:

• Antibody-based proteomics with transcriptomics: Correlate PEBP4 protein levels (detected by antibodies) with mRNA expression to identify post-transcriptional regulation mechanisms.

• Spatial transcriptomics with immunohistochemistry: Overlay PEBP4 antibody staining patterns with spatial transcriptomics data to correlate PEBP4 expression with transcriptional microenvironments.

• Phosphoproteomics with PEBP4 interaction studies: Combine PEBP4 co-immunoprecipitation with phosphoproteomic analysis to identify how PEBP4 influences phosphorylation cascades in signaling networks.

• ChIP-seq with PEBP4 modulation: Investigate how PEBP4 knockdown or overexpression affects transcription factor binding (using ChIP-seq) to identify indirect gene regulatory effects.

• Metabolomics with PEBP4 expression analysis: Correlate PEBP4 antibody-detected expression levels with metabolomic profiles to uncover PEBP4's influence on cellular metabolism.

• Single-cell multi-omics: Integrate PEBP4 antibody-based CyTOF data with single-cell RNA-seq to identify cell populations where PEBP4 protein and mRNA levels correlate or diverge.

• Clinical proteomics: Correlate PEBP4 immunohistochemistry in patient samples with clinical outcomes and treatment responses to identify biomarker potential.

These integrated approaches can help resolve contradictory findings about PEBP4's function in different biological contexts and provide a systems-biology perspective on its role in health and disease.