Phospho-CEBPA (T226) Antibody
Description
Overview of Phospho-CEBPA (T226) Antibody
Phospho-CEBPA (T226) Antibody is a rabbit polyclonal antibody specifically developed to recognize the phosphorylated form of C/EBPα at threonine 226. This antibody is produced using a synthesized peptide derived from human C/EBPα around the phosphorylation site of T226 . The high specificity of this antibody makes it an invaluable tool for studying the regulatory mechanisms of C/EBPα in various biological processes.
The antibody detects endogenous levels of C/EBPα protein only when phosphorylated at T226, providing researchers with the ability to distinguish between the phosphorylated and non-phosphorylated forms of this important transcription factor . This specificity is crucial for understanding how post-translational modifications regulate C/EBPα function in both normal physiology and disease states.
Physical and Chemical Properties
The following table summarizes the key physical and chemical properties of Phospho-CEBPA (T226) Antibody:
| Property | Specification |
|---|---|
| Host | Rabbit |
| Clonality | Polyclonal |
| Immunogen | Synthesized peptide derived from human C/EBPα around the phosphorylation site of T226 |
| Isotype | IgG |
| Form | Liquid in PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide |
| Concentration | 1 mg/ml |
| Storage Recommendation | -20°C or -80°C; avoid repeated freeze-thaw cycles |
| Reactivity | Human, Mouse, Rat, Monkey |
| Applications | Western Blot (WB), ELISA |
| Dilution Recommendations | WB: 1:500-1:2000, ELISA: 1:10000 |
Phosphorylation of CEBPA at T226
Phosphorylation represents a critical post-translational modification that regulates C/EBPα activity. Among the various phosphorylation sites, threonine 226 (T226) has been identified as a significant regulatory site. Research indicates that C/EBPα is a phosphoprotein with multiple phosphorylation sites, including T222, T226, and S230 . These phosphorylation events can significantly alter the protein's function, including its ability to promote or inhibit cellular differentiation.
The biological significance of T226 phosphorylation is still being actively researched, but evidence suggests that this modification may alter C/EBPα's transcriptional activity, DNA binding capacity, and interaction with other proteins. Phosphorylation at this site may represent a mechanism through which various signaling pathways can modulate C/EBPα function in response to cellular stimuli .
Western Blot Analysis
Western blotting represents one of the primary applications for Phospho-CEBPA (T226) Antibody. This technique enables researchers to detect and quantify phosphorylated C/EBPα in cell or tissue lysates. For optimal results, manufacturers typically recommend using the antibody at dilutions ranging from 1:500 to 1:2000 . The antibody specifically recognizes C/EBPα when phosphorylated at T226, producing bands at approximately 30, 42, and 45 kDa depending on the isoform expression in the sample being analyzed .
Proper sample preparation is crucial for successful western blotting with this antibody. Cell lysates should be prepared with phosphatase inhibitors to preserve the phosphorylation state of the protein. Additionally, optimization of blocking conditions, antibody concentrations, and incubation times may be necessary to achieve optimal results with specific sample types .
ELISA Applications
The Phospho-CEBPA (T226) Antibody can also be utilized in Enzyme-Linked Immunosorbent Assays (ELISA) at a recommended dilution of 1:10000 . ELISA provides a quantitative approach for measuring phosphorylated C/EBPα levels in various sample types. This application is particularly valuable for high-throughput screening or when precise quantification of phosphorylated C/EBPα is required.
The high sensitivity of this antibody in ELISA applications makes it suitable for detecting even low levels of phosphorylated C/EBPα, providing researchers with a powerful tool for studying subtle changes in phosphorylation status under various experimental conditions .
Role in Cellular Differentiation
C/EBPα plays a pivotal role in cellular differentiation, particularly in myeloid cells and adipocytes. Research has demonstrated that phosphorylation of C/EBPα can significantly impact its activity in promoting differentiation. Specifically, studies have shown that certain phosphorylation events can inhibit granulopoiesis, the process by which granulocytes develop from hematopoietic stem cells .
While much of the current research has focused on phosphorylation at serine 21 by ERK1/2, the phosphorylation at T226 likely also plays a significant role in modulating C/EBPα's activity in cellular differentiation processes . Understanding these phosphorylation events could provide insights into normal development as well as pathological conditions like acute myeloid leukemia, where C/EBPα function is often disrupted.
Implications in Disease Pathology
Alterations in C/EBPα expression and activity have been implicated in various diseases, particularly in acute myeloid leukemia (AML). Mutations in the CEBPA gene are found in approximately 10-15% of AML cases, highlighting the importance of this transcription factor in normal hematopoiesis .
The phosphorylation status of C/EBPα, including at T226, may have significant implications for disease progression and treatment response. Studies have suggested that phosphorylation can alter the protein's function, potentially contributing to pathological processes . The Phospho-CEBPA (T226) Antibody provides researchers with a tool to investigate these phosphorylation events in disease contexts, potentially leading to new insights into disease mechanisms and therapeutic targets.
Optimization Strategies
For optimal results, several factors may need to be optimized when working with the Phospho-CEBPA (T226) Antibody:
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Antibody dilution: While recommended dilutions are provided (1:500-1:2000 for WB, 1:10000 for ELISA), these may need to be adjusted based on the specific sample type and experimental conditions .
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Incubation conditions: Temperature and duration of primary antibody incubation can significantly impact results and may need optimization.
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Blocking conditions: Different blocking agents and concentrations may be needed to minimize background while maintaining specific signal.
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Sample preparation: Ensuring phosphorylation sites are preserved through the use of phosphatase inhibitors and appropriate lysis conditions is crucial for detecting phosphorylated C/EBPα.
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Detection systems: Various secondary antibodies and detection methods may be employed depending on the sensitivity requirements of the experiment.
Comparative Analysis with Related Antibodies
The Phospho-CEBPA (T226) Antibody is one of several antibodies designed to detect phosphorylated forms of C/EBPα. Another related antibody is the Phospho-C/EBPα (Thr222/226) Antibody, which recognizes C/EBPα when phosphorylated at both threonine 222 and 226 . This dual-specificity antibody provides a different tool for researchers interested in studying multiple phosphorylation events simultaneously.
The choice between these antibodies depends on the specific research question. The Phospho-CEBPA (T226) Antibody offers greater specificity for the T226 phosphorylation site alone, making it valuable for studies focused on this particular modification. In contrast, the dual-specificity antibody may be more appropriate for investigations where both phosphorylation events are of interest .
Product Specs
Target Background
- During monocyte to macrophage differentiation, endosomal/lysosomal proteolytic activity is regulated by cystatin F, whose expression is controlled by the transcription factor C/EBPα. PMID: 30033148
- This study identified 6 frameshift mutations, 1 missense mutation, and 3 synonymous variants in CEBPA. The most common mutation was c.487delG resulting in p.Glu163Ser, observed in 5 cases. Three patients harbored CEBPA double mutations. These findings represent the first screening results of CEBPA gene variants using next-generation sequencing in pediatric acute leukemia patients. PMID: 29947237
- The zinc finger protein ZNF143 binds to the CCCAGCAG site in the CEBPA promoter. PMID: 28900037
- The early stages of adult hepatocellular carcinoma and aggressive pediatric liver cancer share common features, including the conversion of the tumor suppressor C/EBPα into an oncogenic isoform. This transformation leads to the formation of preneoplastic foci where hepatocytes dedifferentiate into cancer cells, ultimately resulting in liver cancer. PMID: 29159818
- Research indicates that upregulation of neuropeptide Y (NPY) inhibits proliferation of adipose-derived stem cells while promoting adipogenesis and upregulating expression of white adipocyte biomarkers, including peroxisome proliferator activated receptor gamma (PPARG), CEBPA, cell death-inducing DFFA-like effector C (CIDEC), and nuclear receptor interacting protein 1 (RIP140). PMID: 28954935
- This study identified for the first time that hepatocyte nuclear factor 4 alpha (HNF4α) and C/EBPα are essential transcriptional regulators for fructose-1,6-bisphosphatase 1 (FBP1) expression in human hepatoma HepG2 cells. PMID: 29566023
- The presence of biallelic CEBPA mutations is a favorable prognostic indicator in acute myeloid leukemia (AML). PMID: 29180507
- C/EBPα mediates anti-inflammatory effects in podocytes. PMID: 27644413
- Proteogenomics profiling studies reveal that activation of C/EBPα, along with the upregulation of its lipogenesis targets, contributes to lipid storage and serves as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC). PMID: 28665611
- Collectively, these findings suggest that the miR-939-Jmjd3 axis disrupts the accessibility of hepatitis B virus enhancer II/core promoter (En II) to essential nuclear factors (C/EBPα and SWI/SNF complex), resulting in compromised viral RNA synthesis and restricted viral multiplication. PMID: 27779233
- Integrating whole-genome sequencing (WGS)-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. PMID: 28031487
- In a large cohort of CEBPAmut AML patients, a high coincidence of GATA2 mutations was observed, particularly within the subgroup of patients with CEBPAbi mutations. PMID: 27375010
- A decision analysis comparing allogeneic hematopoietic cell transplantation (allo-HCT) versus chemotherapy in first complete remission for patients with cytogenetically intermediate-risk AML, considering the presence or absence of FLT3-ITD, NPM1, and CEBPA mutations, revealed that allo-HCT was a preferred postremission strategy in patients with FLT3-ITD, while chemotherapy was favored in patients with biallelic CEBPA mutations. PMID: 27040395
- This study provides the first evidence that the c.690G>T, p.(Thr230Thr) (rs34529039) polymorphism of the CEBPA gene, along with upregulation of its mRNA expression, are negative factors that worsen ovarian cancer outcomes. PMID: 27602952
- CSF3R mutations occur in conjunction with CEBPA mutations in pediatric AML. PMID: 27143256
- While extensive research exists on how C/EBPα orchestrates granulopoiesis, understanding the molecular transformation events, the roles of cooperating mutations, and clonal evolution during C/EBPα deregulation in leukemia remains unclear. This review summarizes recent findings related to these topics, with a focus on CEBPA mutations. PMID: 28720765
- miR-182 is a potent regulator of C/EBPα. A regulatory loop exists between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression through direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein levels and impairs granulopoiesis in vitro and in vivo. PMID: 28663557
- CCAAT/enhancer-binding protein homologous protein (CHOP) negatively regulates Polo-like kinase 2 (PLK2) expression by recruiting C/EBPα to the upstream promoter in human osteosarcoma cell lines during endoplasmic reticulum (ER) stress. PMID: 28652211
- C/EBPα overexpression suppressed epithelial-mesenchymal transition (EMT), characterized by a gain of epithelial markers and a loss of mesenchymal markers. Further studies revealed that C/EBPα suppressed the transcription of β-catenin and downregulated the levels of its downstream targets. PMID: 28746919
- Binding of C/EBPα was associated with increased deacetylation near the transcription start site (TSS) of the PLK1 promoter. PMID: 28341486
- A correlation between myocyte enhancer factor 2C (MEF2C) and CEBPA was observed in chronic myeloid leukemia (CML) disease progression. PMID: 27297623
- CEBPA gene expression is significantly associated with long-term changes in blood pressure, establishing a link between gene expression and blood pressure. PMID: 28784648
- This study provides evidence that CCAAT/enhancer-binding protein alpha directly binds to the miR-203 gene within its hairpin region, thereby inducing miR-203 transcription. PMID: 28640877
- High CEBP expression is associated with glioblastomas. PMID: 27591677
- This study identified high frequencies of mutations in CEBPA (32.7%), GATA2 (22.4%), NPM1 (15.5%), SETBP1 (12.1%), and U2AF1. PMID: 27389056
- The study demonstrates that excess p30 cooperated with tribbles 2 (TRIB2) only in the presence of p42 to accelerate AML, and the direct interaction and degradation of C/EBPa p42 are essential for TRIB2-mediated AML. PMID: 26996668
- A single +42-kb enhancer is crucial for CEBPA expression specifically in myeloid cells. PMID: 26966090
- This study reveals the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors. These findings pave the way for personalized clinical trials for this disease. PMID: 27034432
- This study established a reliable and straightforward screening method based on the multidimensional analysis of readily available phenotypic parameters. This method is suitable for large-scale detection of CEBPA-dm status and has the potential to overcome technical challenges associated with molecular methods. PMID: 28250006
- This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-alpha to bind DNA and reduces transactivation, leading to AML. PMID: 26721895
- SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPβ-LIP. This process is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5' untranslated regions (5' UTRs) of both mRNAs. PMID: 26762974
- SHP2-ERK2 signaling acts upstream of C/EBPα as a regulator of cell surface I antigen synthesis. PMID: 27600951
- This study highlights the significance of C/EBPα for neutrophil maturation, its role in myeloid priming of hematopoietic stem and progenitor cells, and its indispensable requirement for AML development. PMID: 28179278
- This study found significantly higher frequencies for NPM1-mutated (24.2%) and CEBPA-mutated (12.1%). PMID: 27436336
- These findings demonstrate that low-level expression of the human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by C/EBP transcription factors in monocytic cells. This suggests that lowly expressed ACAT2 catalyzes the synthesis of certain cholesterol esters (CE/SE) that are assembled into lipoproteins for secretion. PMID: 27688151
- This study highlighted two novel promoter Krüppel-like factor 1 (KLF1) and 3'-region C/EBPα motifs in the phenylalanine hydroxylase (PAH) gene, which decrease transcription in vitro and, therefore, could be considered as PAH expression modifiers. PMID: 27447460
- The QA repeat domain of TCERG1 is required for the relocalization of CEBPα. PMID: 26264132
- No mutations were detected in the CCAAT/enhancer binding protein alpha gene, but a known polymorphism (c.584_589dupACCCGC) was observed in 26 (28.3%) patients. PMID: 25932436
- The p53-KLF4-CEBPA axis is dysregulated in AML but can be functionally restored by conventional chemotherapy and novel p53-activating treatments. PMID: 26408402
- C/EBPα inhibited breast cancer cell growth via a novel miR-134/CREB signaling pathway. PMID: 26823765
- C/EBP-alpha was predominantly expressed in hepatocytes in normal liver, but its expression decreased significantly in liver fibrosis. PMID: 26722507
- The efficient repression of E2F-dependent S-phase genes and the activation of differentiation genes depend on the balanced DNA binding capacity of C/EBPα. PMID: 27131901
- This is the first report identifying miR-381 as a suppressor of C/EBPα-dependent Cx43 expression in breast cancer cells. The miR-381-C/EBPα-Cx43 axis may serve as a useful diagnostic and therapeutic target for metastatic breast cancer. PMID: 26450928
- These findings suggest that C/EBPα-saRNA successfully inhibited HCC metastasis by inhibiting EGFR/β-catenin signaling pathway-mediated EMT in vitro and in vivo. PMID: 27050434
- These results suggest that a genetic predisposition to higher IL-6 production is associated with an increased risk of HBV infection and hepatic inflammation, potentially due to the regulatory effect of C/EBPα on Th17 and Treg responses. PMID: 26447433
- This study demonstrated that suppression of C/EBPa P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and all-trans retinoic acid (ATRA) sensitivity of acute promyelocytic leukemia cells. PMID: 26397153
- This research identifies GCN5 as a negative regulator of C/EBPα and demonstrates the importance of C/EBPα acetylation in myeloid differentiation. PMID: 27005833
- These findings suggest that younger age, the presence of mirror repeats, and a high CEBPA expression level in relation to potential topoisomerase II (topo II) sites might influence the incidence of B-ZIP in-frame copy number variations (CNVs) through aberrant recombination-mediated DNA repair mechanisms. PMID: 26460249
- Reprogramming human B cells into induced pluripotent stem cells is enhanced by C/EBPa. PMID: 26500142
- This is the first report on the regulation mechanism of SIRT7 gene, where histone deacetylase 3 (HDAC3) collaborates with C/EBPα to occupy its responding element in the upstream region of the SIRT7 gene and repressed its expression in human cells. PMID: 26704017
Q&A
Primary Applications and Functional Relevance
Q: What is the primary function of the Phospho-CEBPA (T226) Antibody in academic research?
The Phospho-CEBPA (T226) Antibody is a critical tool for studying CCAAT/enhancer-binding protein alpha (CEBPA) phosphorylation at threonine 226, a modification linked to metabolic regulation and myeloid cell differentiation. It is used to detect phosphorylated CEBPA in pathways such as gluconeogenesis, lipogenesis, and granulopoiesis. Key applications include:
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Western blotting (WB): Detecting phosphorylation under fasting or insulin signaling conditions .
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ELISA: Quantifying phosphorylation levels in biological samples .
Q: How does phosphorylation at T226 regulate CEBPA activity?
Phosphorylation at T226 is part of the CR4 domain, which undergoes dephosphorylation during fasting. This dephosphorylation upregulates gluconeogenic genes like G6Pase and PEPCK, contributing to hepatic glucose production . In contrast, phosphorylation at S21 (via ERK1/2) induces conformational changes that inhibit granulopoiesis by disrupting CEBPA dimerization .
Experimental Design and Cross-Reactivity
Q: How does the antibody’s cross-reactivity across species influence experimental design?
The antibody reacts with human, mouse, rat, and monkey CEBPA . This broad reactivity enables comparative studies but requires careful validation. For example:
Q: What controls are essential for validating Phospho-CEBPA (T226) Antibody specificity?
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Peptide blocking: Use a non-phosphorylated peptide (AA range: 192–241) to confirm signal specificity .
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Phosphatase treatment: Incubate lysates with λ-phosphatase to demonstrate loss of signal .
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Negative controls: Include samples from unstimulated or non-target tissues (e.g., non-hepatic cells) .
Troubleshooting and Data Interpretation
Q: How to address inconsistent Western blot results for Phospho-CEBPA (T226)?
Common issues and solutions include:
Q: What explains conflicting data on CEBPA phosphorylation in metabolic vs. myeloid studies?
Phosphorylation at distinct sites (e.g., T226 vs. S21) regulates separate pathways:
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S21: Regulates granulopoiesis by altering CEBPA dimerization .
Conflicts arise from site-specific effects, requiring antibodies targeting individual residues.
Advanced Research Applications
Q: How to integrate Phospho-CEBPA (T226) data with metabolic flux analysis?
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Sample preparation: Use hepatic lysates from fasted mice to induce T226 dephosphorylation .
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Parallel assays: Measure G6Pase and PEPCK mRNA alongside phosphorylation levels.
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Statistical analysis: Correlate CEBPA phosphorylation with glucose output rates using linear regression.
Q: What experimental models are optimal for studying CEBPA T226 phosphorylation?
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In vivo: Mouse knock-in mutants (TTS mice) with alanine substitutions at T222/T226/S230 to study gluconeogenesis .
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In vitro: Hepatocytes treated with insulin/PP2A inhibitors to mimic dephosphorylation .
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Cell lines: COS7 or 293T cells transfected with CEBPA mutants for mechanistic studies .
Methodological Insights
Q: What protocols optimize Phospho-CEBPA (T226) detection in low-abundance samples?
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Lysis buffer: Include phosphatase inhibitors (e.g., NaF, β-glycerophosphate) to preserve phosphorylation .
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WB conditions: Use PVDF membranes and prolonged transfer times (2–3 hours) for large proteins (>30 kDa) .
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ELISA: Pre-clear samples with non-phosphorylated peptides to reduce non-specific binding .
Q: How to reconcile Phospho-CEBPA (T226) findings with CEBPA’s role as a tumor suppressor?
Phosphorylation at T226 may modulate CEBPA’s transcriptional activity in cancer contexts. For example:
