Phospho-CEBPE (T74) Antibody
Product Specs
Target Background
- CEBPE expression was highest in multipotent progenitor cells (S1) and declined sharply as cells progressed to B-cell-committed progenitors, including pre-B-I cells (S2), pre-B-II cells (S3), and immature B cells (S4). PMID: 26437776
- Research has demonstrated that loss-of-function mutations in both CEBPE and SMARCD2, identified in patients with neutrophil-specific granule deficiency (SGD), disrupt the interaction with SWI/SNF and lead to the suppression of secondary granule gene expression. This provides a molecular basis for the pathogenesis of SGD. PMID: 28369034
- PML/RARalpha acts synergistically with C/EBPepsilon to reactivate the C/EBPepsilon target gene G0S2. This contributes to the differentiation of acute promyelocytic leukemia cells induced by all-trans retinoic acid (ATRA) and potentially contributes to clinical remission. PMID: 27605212
- The rs7088318 (PIP4K2A) and rs2239633 (CEBPE) polymorphisms were not found to be associated with acute lymphoblastic leukemia (ALL) risk. PMID: 28476190
- The minor allele of the CEBPE variant, associated with lower basophil count, has been previously linked to Amerindian ancestry and a higher risk of ALL in Hispanic populations. PMID: 28158719
- The rs45496295 (C > T) polymorphism in the heterozygous state was identified in 73.9% of beta-thalassemia intermedia patients. PMID: 27829304
- Variants within IKZF1, ARID5B, and CEBPE were associated with increased risk of pediatric ALL. PMID: 27184773
- Genotypic and allelic frequencies differed significantly between cases and controls at IKZF1-rs4132601 (p=0.039, p=0.015) and ARID5B-rs10821936 (p=0.028, p=0.026). PMID: 27644650
- Variants within IKZF1, ARID5B, and CEBPE were associated with increased ALL risk, with the effects for ARID5B and CEBPE being most prominent in the high-hyperdiploid ALL subtype within the California Hispanic population. PMID: 25761407
- Research indicates no significant association of transcription factors rs4132601 (IKZF1), rs7089424 (ARID5B), and rs2239633 (CEBPE) with the risk of pediatric non-Hodgkin lymphoma (NHL). PMID: 25005032
- A study revealed that the CEBPE rs2239633 variant is associated with a decreased risk of childhood B-cell ALL in European populations. PMID: 25938438
- A novel in-frame deletion in the leucine zipper domain of CEBPE has been identified as a causative factor in neutrophil-specific granule deficiency. PMID: 26019275
- During neutrophil development, acetylation of lysines 121 and 198 has been found to be crucial for terminal neutrophil differentiation and the expression of neutrophil-specific granule proteins, including lactoferrin and collagenase. PMID: 25568349
- Studies have shown that the CCAAT/enhancer binding protein-epsilon (CEBPE) rs2239633 polymorphism was significantly associated with the risk of childhood acute lymphoblastic leukemia (ALL). PMID: 25195121
- A study found that previously identified childhood ALL susceptibility loci in ARID5B and CEBPE exhibit consistent risk effects across both Hispanic and non-Hispanic White populations, providing strong supportive evidence for susceptibility at these loci. PMID: 23836053
- Germline variants in IKZF1, ARID5B, and CEBPE have been identified as risk factors for adult-onset acute lymphoblastic leukemia, as analyzed by the GMALL study group. PMID: 24497567
- miR-130a plays a crucial role in regulating the timed expression of C/EBP-epsilon during granulopoiesis. PMID: 24398327
- The rs2239632 polymorphism may regulate the expression of the CEBPE gene. Its risk allele (G) might increase the gene product and contribute to leukemogenesis. Individuals carrying this allele or the corresponding haplotype might have increased susceptibility to ALL. PMID: 23719191
- rs4132601 in IKZF1 and rs2239633 in CEBPE were not found to be significantly related to childhood ALL. PMID: 23608171
- A case study provides the first evidence that chromosome duplication and cryptic insertion produce the CEBPE-IGH fusion and that multiple CEBPE-IGH recombinations can occur within a leukemic cell. PMID: 22137487
- Collectively, research findings demonstrate that C/EBPepsilon participates in the induction of PI3Kgamma by all-trans retinoic acid. PMID: 20661648
- CEBPE has been identified as a causative agent in the development of Neutrophil-specific granule deficiency. PMID: 11753076
- CEBPE interacts with other transcription factors to regulate the transcription of the gene encoding eosinophil granule major basic protein. PMID: 12202480
- C/EBP epsilon is both essential and sufficient for the expression of a specific subset of neutrophil secondary granule genes. PMID: 12515729
- Upon induction of maturation, C/EBP epsilon binds to the lactoferrin (LF) promoter, which correlates with LF expression. PMID: 12522000
- Overexpression of C/EBPepsilon in myeloid cells leads to down-regulation of c-Myc. PMID: 12947005
- Retinoic acid signaling during granulocytic differentiation involves regulated expression of CHOP protein and C/EBPepsilon in a coordinated manner. PMID: 15308577
- Research suggests that ATRA-induced regulation of Stat2, ICSBP, and C/EBPepsilon is dependent on active Stat1, and that failure to correctly regulate these transcription factors is associated with the inhibition of monocytic differentiation. PMID: 16918696
- Decreased Gfi-1 levels in an SGD patient, coupled with the mutant C/EBPepsilon, block the expression of secondary granules proteins, contributing to the underlying etiology of the disease in this patient. PMID: 17244686
- The interaction of the activated NFkappaB pathway and C/EBP-epsilon may be crucial in the selective activation of a subset of C/EBP-epsilon-responsive genes. PMID: 17255362
- The type IV isoform of PML interacts with PU.1, promotes its association with p300, and subsequently enhances PU.1-induced transcription and granulocytic differentiation. Furthermore, PU.1 directly activates the transcription of the C/EBPepsilon gene. PMID: 17562868
- C/EBP epsilon is a critical transcription factor for tumor necrosis factor alpha-induced up-regulation of phospholipid hydroperoxide glutathione peroxidase (PHGPx) expression in non-differentiated HL60 cells. PMID: 17688422
- C/EBP- isoforms can reprogram myeloid lineage commitment and differentiation, consistent with their predicted activities based on activator and repressor domains and in vitro functional activities. PMID: 18832658
- Significant quantities of C-EBPepsilon are expressed in human neutrophils, especially the p14 and p32 variants. PMID: 19109189
Q&A
Antibody Specificity and Validation
Q: How can I confirm the antibody’s specificity for phosphorylated T74 residues in CEBPE?
To validate phosphorylation-specific binding, perform peptide blocking experiments using the phosphopeptide immunogen. For example, Abcam’s product (ab192806) demonstrates a loss of signal when the antigen-specific peptide is added to HUVEC cell lysates treated with UV . Similarly, Boster Bio recommends blocking with a phosphopeptide to eliminate non-specific signals in immunofluorescence and IHC .
Table 1: Recommended Blocking Peptide Strategies
Q: Are cross-reactivity issues reported for this antibody?
Some vendors note predicted reactivity with rat and mouse based on sequence homology (e.g., St John’s Labs) , but human-specific validation dominates. Confirm species compatibility using negative controls (e.g., lysates from non-human cell lines).
Experimental Design and Optimization
Q: What dilution ranges should I use for different applications?
Optimal dilutions vary by technique:
Q: How should I handle phosphorylation-dependent detection in heterogeneous cell populations?
Use phosphatase inhibitors (e.g., sodium orthovanadate) during sample preparation to preserve phosphorylation states. For dynamic phosphorylation studies, synchronize stimuli (e.g., UV treatment) and harvest cells at timed intervals to capture transient modifications .
Troubleshooting and Data Interpretation
Q: What causes non-specific bands in WB using this antibody?
Potential sources include:
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Cross-reactivity: Non-phosphorylated CEBPE isoforms or homologs (e.g., C/EBPα/β) .
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Protein degradation: Incomplete inhibition of proteases during lysate preparation.
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Antibody aggregation: Repeated freeze-thaw cycles (mitigate by aliquoting) .
Q: How can I resolve discrepancies between WB and ELISA results?
Compare epitope accessibility:
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WB: Denaturing conditions may expose epitopes masked in native states.
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ELISA: Requires conformational integrity for binding. Use both methods to confirm phosphorylation status in complementary contexts .
Advanced Research Applications
Q: Can this antibody be used to study CEBPE phosphorylation in myeloid differentiation?
Yes. CEBPE is critical for promyelocyte-myelocyte transition . Design experiments with granulocyte colony-stimulating factor (G-CSF) or all-trans retinoic acid (ATRA) to induce differentiation. Use flow cytometry to isolate cell subsets (e.g., CD11b+HLA-DR− myeloblasts) for WB analysis .
Q: How can I quantify phosphorylation levels using phospho-specific ELISA?
Generate a standard curve using recombinant phospho-CEBPE protein. Normalize values to total CEBPE levels (via non-phospho-specific ELISA) to account for protein abundance variability .
Methodological Considerations
Q: What controls should I include to validate phosphorylation-specific signals?
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Negative control: Cells treated with a kinase inhibitor (e.g., staurosporine) to reduce T74 phosphorylation.
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Positive control: Cells stimulated with UV radiation or cytokines (e.g., IL-6) known to induce CEBPE phosphorylation .
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Peptide competition: As described in Table 1.
Q: How does antibody affinity affect detection sensitivity?
High-affinity antibodies enable lower dilutions (e.g., 1:3000 for WB) , but may require extended primary incubation times. Use affinity-purified antibodies (e.g., Abcam, Boster) to minimize background .
Species and Cross-Reactivity
Q: Is the antibody suitable for rodent models?
While some vendors predict reactivity with rat/mouse (e.g., St John’s Labs) , human-specific validation dominates. Perform pilot studies with murine cell lines (e.g., RAW264.7 macrophages) to confirm cross-reactivity.
