Phospho-CEBPA (Thr230) Antibody
Description
Western Blot Validation
-
Boster Bio (A00386T230): Demonstrated specificity for phosphorylated CEBPA in nuclear extracts of HepG2 cells using a Cytoplasmic and Nuclear Fractionation kit. WB dilution recommendations: 1:500–1:2000 .
-
Abbexa (ABB0014): Tested for WB in human, mouse, and rat lysates, with optimal dilutions determined by end-users .
Cross-Reactivity
Neither antibody cross-reacts with eNOS or nNOS, ensuring specificity for Thr230-phosphorylated CEBPA .
Role in Transcriptional Regulation
CEBPA phosphorylation at Thr230 is linked to its activation in processes like adipogenesis and granulopoiesis. The antibody enables detection of this modification, which correlates with transcriptional activity . For example:
-
Adipogenesis: CEBPA Thr230 phosphorylation enhances its binding to promoters of genes like ADIPOQ, promoting adipocyte differentiation .
-
Gluconeogenesis: In liver tissue, phosphorylated CEBPA cooperates with FOXO1 to activate genes such as PCK1 and G6PC1 .
Prognostic Significance in Acute Myeloid Leukemia (AML)
Research highlights CEBPA mutations and epigenetic silencing as critical prognostic markers in AML :
-
Double Mutations: Patients with CEBPA double mutations exhibit favorable survival outcomes (hazard ratio 0.23, P=0.04) .
-
Silenced CEBPA: Epigenetic silencing via promoter hypermethylation correlates with poor prognosis and relapse in pediatric AML .
The antibody’s ability to detect Thr230 phosphorylation could complement these studies by identifying active CEBPA states in AML samples.
Experimental Considerations
Product Specs
Target Background
- During the differentiation of monocytes into macrophages, the endosomal/lysosomal proteolytic activity is regulated by cystatin F, whose expression is under the control of the transcriptional factor C/EBP alpha. PMID: 30033148
- Our research identified 6 frameshift mutations, 1 missense mutation, and 3 synonymous variants in the CEBPA gene. The most prevalent mutation was c.487del G, resulting in p.Glu163Ser, observed in 5 cases. Three patients exhibited double CEBPA mutations. In conclusion, the variants identified in this study represent the initial screening results of these genes using next-generation sequencing in pediatric acute leukemia patients. PMID: 29947237
- The zinc finger protein ZNF143 binds to the CCCAGCAG site within the CEBPA promoter. PMID: 28900037
- The initial stages of both adult hepatocellular carcinoma and aggressive pediatric liver cancer share identical characteristics, including the conversion of the tumor suppressor C/EBPalpha into an oncogenic isoform. This conversion further creates preneoplastic foci where hepatocytes dedifferentiate into cancer cells, leading to the development of liver cancer. PMID: 29159818
- Data suggests that upregulation of neuropeptide Y (NPY) inhibits proliferation of adipose-derived stem cells while promoting adipogenesis and upregulating the expression of white adipocyte biomarkers such as peroxisome proliferator activated receptor gamma (PPARG), CEBPA, cell death-inducing DFFA-like effector C (CIDEC), and nuclear receptor interacting protein 1 (RIP140). PMID: 28954935
- Our study identified for the first time that hepatocyte nuclear factor 4 alpha (HNF4alpha) and C/EBPalpha are crucial transcriptional regulators for the expression of fructose-1,6-bisphosphatase 1 (FBP1) 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/EBP-alpha mediates anti-inflammatory effects in podocytes. PMID: 27644413
- Proteogenomics profiling studies reveal that activation of C/EBPalpha, along with the upregulation of its lipogenesis targets, accounts for lipid storage and serves as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC). PMID: 28665611
- Collectively, our findings suggest that the miR-939-Jmjd3 axis disrupts the accessibility of the hepatitis B virus enhancer II/core promoter (En II) promoter to essential nuclear factors, such as C/EBPalpha and the SWI/SNF complex. This disruption leads to compromised viral RNA synthesis and, consequently, restricted viral multiplication. PMID: 27779233
- The integration of 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 our study of a large cohort of CEBPAmut AML patients, we observed a high coincidence of GATA2mut, 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 acute myeloid leukemia, depending on the presence or absence of FLT3-ITD, NPM1, and CEBPA mutations, indicated that allo-HCT was a preferred postremission strategy for patients with FLT3-ITD, whereas chemotherapy was favored for patients with biallelic CEBPA mutations. PMID: 27040395
- This is the first study to provide evidence that the c.690G>T, p.(Thr230Thr) (rs34529039) polymorphism of the CEBPA gene, coupled with upregulation of its mRNA expression, are negative factors associated with worsening ovarian cancer outcome. PMID: 27602952
- CSF3R mutations co-occur with CEBPA mutations in pediatric acute myeloid leukemia. PMID: 27143256
- While a significant amount is known about how C/EBPalpha orchestrates granulopoiesis, our understanding of molecular transformation events, the role(s) of cooperating mutations, and clonal evolution during C/EBPalpha deregulation in leukemia remains unclear. This review summarizes the latest research addressing these topics, with a particular emphasis on CEBPA mutations. PMID: 28720765
- miR-182 is a potent regulator of C/EBPalpha. A regulatory loop exists between C/EBPalpha and miR-182. While C/EBPalpha blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPalpha protein levels and impairs granulopoiesis both in vitro and in vivo. PMID: 28663557
- CHOP negatively regulates Polo-like kinase 2 (PLK2) expression by recruiting C/EBPalpha to the upstream promoter in human osteosarcoma cell lines during endoplasmic reticulum (ER) stress. PMID: 28652211
- C/EBPalpha overexpression suppressed the epithelial-mesenchymal transition (EMT), characterized by a gain of epithelial and loss of mesenchymal markers. Further investigation showed that C/EBPalpha suppressed the transcription of beta-catenin and downregulated the levels of its downstream targets. PMID: 28746919
- The binding of C/EBPalpha was associated with increased deacetylation near the transcription start site (TSS) of the PLK1 promoter. PMID: 28341486
- A correlation between 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, providing a link between gene expression and blood pressure. PMID: 28784648
- We provide evidence that CCAAT/enhancer-binding protein alpha directly binds the miR-203 gene within its hairpin region, thereby inducing miR-203 transcription. PMID: 28640877
- High CEBP expression is associated with glioblastomas. PMID: 27591677
- Our study identified high frequencies of mutations in CEBPA (32.7%), GATA2 (22.4%), NPM1 (15.5%), SETBP1 (12.1%), and U2AF1. PMID: 27389056
- Our data show that excess p30 cooperated with TRIB2 only in the presence of p42 to accelerate acute myeloid leukemia (AML), and the direct interaction and degradation of C/EBPa p42 is required for TRIB2-mediated AML. PMID: 26996668
- A single +42-kb enhancer is essential for CEBPA expression specifically in myeloid cells. PMID: 26966090
- The co-occurrence of mutations in CSF3R and CEBPA in a well-defined subset of acute myeloid leukemia, which uniformly responds to JAK inhibitors, paves the way for personalized clinical trials for this disease. PMID: 27034432
- We established a reliable and straightforward screening method, based simply on the multidimensional analysis of widely available phenotypic parameters, suitable for large-scale detection of CEBPA-dm status and potentially capable of overcoming technical issues related to 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 acute myeloid leukemia. PMID: 26721895
- SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPalpha-p30 and C/EBPbeta-LIP, which 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/EBPalpha as a regulator of cell surface I antigen synthesis. PMID: 27600951
- The importance of C/EBPalpha for neutrophil maturation, its role in myeloid priming of hematopoietic stem and progenitor cells, and its indispensable requirement for AML development have been highlighted. PMID: 28179278
- Our findings demonstrate significantly higher frequencies for NPM1-mutated (24.2%) and CEBPA-mutated (12.1%). PMID: 27436336
- Results demonstrate that the low-level expression of the human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by the C/EBP transcription factors in monocytic cells. This suggests that lowly expressed ACAT2 catalyzes the synthesis of certain cholesterol esters (CE)/sterol esters (SE) that are assembled into lipoproteins for secretion. PMID: 27688151
- Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the phenylalanine hydroxylase (PAH) gene, which decrease transcription in vitro. Therefore, these motifs could be considered as PAH expression modifiers. PMID: 27447460
- The QA repeat domain of TCERG1 is required for the relocalization of CEBPalpha. PMID: 26264132
- No mutations were detected in the CCAAT/enhancer binding protein alpha gene, but a known polymorphism (c.584_589dup ACCCGC) was observed in 26 (28.3%) patients. PMID: 25932436
- The p53-KLF4-CEBPA axis is deregulated in AML but can be functionally restored by conventional chemotherapy and novel p53 activating treatments. PMID: 26408402
- C/EBPalpha inhibited breast cancer cell growth via a novel miR-134/CREB signaling pathway. PMID: 26823765
- C/EBP-alpha was primarily 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 reside in the balanced DNA binding capacity of C/EBP alpha. PMID: 27131901
- We are the first to identify that miR-381 suppresses C/EBPalpha-dependent Cx43 expression in breast cancer cells. The miR-381-C/EBPalpha-Cx43 axis might be a valuable diagnostic and therapeutic target for metastatic breast cancer. PMID: 26450928
- Results suggested that C/EBPalpha-saRNA successfully inhibited HCC metastasis by inhibiting EGFR/beta-catenin signaling pathway-mediated EMT in vitro and in vivo. PMID: 27050434
- These findings suggest that genetic predisposition to higher IL-6 production is associated with an increased risk of HBV infection and hepatic inflammation, which might be attributed to C/EBPalpha-mediated regulatory effects on Th17 and Treg responses. PMID: 26447433
- The present 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
- Data uncover GCN5 as a negative regulator of C/EBPalpha and demonstrate the importance of C/EBPalpha acetylation in myeloid differentiation. PMID: 27005833
- Younger age, the presence of mirror repeats, and high CEBPA expression levels in relation to potential topo II-sites might affect 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 the SIRT7 gene, in which HDAC3 collaborated with C/EBPalpha to occupy its responding element in the upstream region of the SIRT7 gene and repressed its expression in human cells. PMID: 26704017
Q&A
What is Phospho-CEBPA (Thr230) Antibody and what is its specificity?
Phospho-CEBPA (Thr230) Antibody is a rabbit polyclonal antibody specifically designed to detect CCAAT/enhancer-binding protein alpha (C/EBPα) only when phosphorylated at threonine 230. The antibody binds to endogenous CEBPA at the amino acid region 170-250 exclusively when the Thr230 residue is phosphorylated . The specificity is achieved through affinity purification techniques, where non-phospho-specific antibodies are removed by chromatography using non-phosphopeptide . The immunogen used for antibody production is a synthesized phospho-peptide surrounding the phosphorylation site of human C/EBP Alpha (phospho Thr230) .
What experimental applications is Phospho-CEBPA (Thr230) Antibody suitable for?
Based on the available data, Phospho-CEBPA (Thr230) Antibody is validated for:
While some related phospho-CEBPA antibodies are indicated for additional applications such as immunohistochemistry (IHC) and immunofluorescence (IF), the Thr230-specific antibody has primarily been validated for WB and ELISA applications .
What species reactivity has been confirmed for this antibody?
The Phospho-CEBPA (Thr230) Antibody has been validated to react with:
-
Human
-
Mouse
-
Rat
This cross-species reactivity makes the antibody valuable for comparative studies across multiple model systems .
What is the optimal storage condition for maintaining antibody functionality?
For maximum stability and activity retention, Phospho-CEBPA (Thr230) Antibody should be stored at -20°C for up to 1 year from the date of receipt . The antibody is typically formulated in PBS with 50% glycerol, 0.5% BSA and 0.02% sodium azide or similar stabilizing buffers . It's important to avoid repeated freeze-thaw cycles which can compromise antibody functionality and specificity.
What is the recommended protocol for Western blot detection of phosphorylated CEBPA?
For optimal Western blot results with Phospho-CEBPA (Thr230) Antibody:
-
Extract proteins from cells of interest. Nuclear extraction is recommended as CEBPA is a nuclear transcription factor (as demonstrated in HepG2 cells where nuclear extraction was performed using a Cytoplasmic and Nuclear Fractionation kit) .
-
Separate proteins by SDS-PAGE and transfer to a membrane.
-
Block the membrane using standard blocking buffer.
-
Dilute the Phospho-CEBPA (Thr230) Antibody at 1:500-1:2000 in appropriate antibody dilution buffer .
-
Incubate the membrane with diluted primary antibody overnight at 4°C or for 2 hours at room temperature.
-
Wash the membrane thoroughly with TBST or similar wash buffer.
-
Incubate with an appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG).
-
Develop using standard chemiluminescence detection methods.
Expected bands appear at approximately 30-45 kDa, with specific isoforms at 42 and 45 kDa corresponding to different translation products of CEBPA .
How can I implement an ELISA protocol using this antibody?
For ELISA applications with Phospho-CEBPA (Thr230) Antibody:
-
Coat the ELISA plate with capture antibody or target protein.
-
Block non-specific binding sites with an appropriate blocking buffer.
-
Add samples containing phosphorylated CEBPA.
-
Dilute Phospho-CEBPA (Thr230) Antibody at 1:10000 for optimal detection sensitivity .
-
Add diluted antibody to the wells and incubate according to your established ELISA protocol.
-
Wash thoroughly to remove unbound antibody.
-
Add appropriate HRP-conjugated secondary antibody.
-
Develop using a suitable substrate and measure absorbance.
For cell-based ELISA applications, researchers can follow protocols similar to those established for related phospho-CEBP antibodies, such as the C/EBP-beta Phospho-Thr235/188 Colorimetric Cell-Based ELISA protocol .
What controls should be included when working with phospho-specific antibodies?
When working with Phospho-CEBPA (Thr230) Antibody, include the following controls:
-
Antigen-specific peptide control: Treatment with an antigen-specific peptide should abolish the signal, confirming antibody specificity (as demonstrated in Western blot analysis of cos-7 cells) .
-
Dephosphorylation control: Treat a portion of your samples with phosphatase to demonstrate that signal loss correlates with dephosphorylation.
-
Total CEBPA control: Run parallel samples with a total CEBPA antibody to compare phosphorylated versus total protein levels.
-
Positive control: Include samples known to have high levels of phosphorylated CEBPA at Thr230, such as HepG2 cells or cos-7 cells .
-
Loading control: Include detection of housekeeping proteins like GAPDH to ensure equal loading across samples.
What is the expected molecular weight pattern for phosphorylated CEBPA?
CEBPA is detected at multiple molecular weights due to alternative translation initiation sites:
-
42-45 kDa: Full-length CEBPA protein
-
30 kDa: Truncated isoform
The phospho-specific antibody should detect bands at these molecular weights when the Thr230 site is phosphorylated . The presence of multiple bands is expected and reflects the biology of CEBPA translation, where alternative in-frame non-AUG (GUG) and AUG start codons result in protein isoforms with different lengths .
How can I improve sensitivity when signals are weak in Western blotting?
If experiencing weak signals with Phospho-CEBPA (Thr230) Antibody:
-
Optimize protein extraction: Ensure efficient nuclear extraction as CEBPA is a nuclear protein. The data shows successful detection using nuclear extracts from HepG2 cells .
-
Increase protein concentration: Load more total protein if phosphorylated CEBPA is expressed at low levels.
-
Optimize antibody concentration: Try the higher end of the recommended dilution range (1:500 instead of 1:2000) .
-
Extended incubation: Increase primary antibody incubation time to overnight at 4°C.
-
Enhanced detection systems: Use high-sensitivity chemiluminescent substrates or signal amplification methods.
-
Phosphatase inhibitors: Ensure your lysis buffer contains adequate phosphatase inhibitors to preserve phosphorylation status.
How do I distinguish between specific and non-specific signals?
To confidently identify specific phospho-CEBPA (Thr230) signals:
-
Pre-absorption control: Treatment with the immunizing phosphopeptide should eliminate specific bands. Data shows this control effectively eliminated signal in cos-7 cell extracts .
-
Molecular weight verification: Specific signals should appear at the expected molecular weights (30, 42, and 45 kDa) .
-
Compare with total CEBPA antibody: The phospho-specific and total antibodies should detect proteins of the same molecular weight, though relative intensities may differ based on phosphorylation status.
-
Positive control samples: Include samples with known phosphorylation status of CEBPA at Thr230.
How does phosphorylation at Thr230 compare with other CEBPA phosphorylation sites?
CEBPA is regulated by multiple phosphorylation events. The search results indicate several phosphorylation sites that are commercially targeted by antibodies:
Each phosphorylation site affects CEBPA function differently. While the search results don't provide specific functional differences between these sites, researchers should consider that:
-
The phosphorylation at Thr230 likely affects a different aspect of CEBPA function than phosphorylation at other sites.
-
Multiple phosphorylation events may work in concert or antagonistically.
-
Different kinases and signaling pathways may regulate each phosphorylation site.
For comprehensive functional studies, researchers should consider analyzing multiple phosphorylation sites simultaneously.
What is the relationship between CEBPA and CEBPB phosphorylation?
The search results indicate related antibodies for both CEBPA and CEBPB phosphorylation:
This suggests potential parallel regulation of these transcription factors. Researchers interested in CEBP family regulation should consider:
-
CEBPA and CEBPB can form heterodimers , and their phosphorylation may affect dimerization.
-
Similar kinases may target corresponding phosphorylation sites on both proteins.
-
Coordinated phosphorylation may regulate their combined activity in gene expression.
Experiments comparing the phosphorylation status of both proteins under various conditions would provide insight into their coordinated regulation.
What experimental approaches can investigate the functional consequences of CEBPA Thr230 phosphorylation?
To study the functional impact of CEBPA Thr230 phosphorylation:
-
Phosphomimetic and phospho-deficient mutants: Generate CEBPA with T230E (phosphomimetic) or T230A (phospho-deficient) mutations to study the effects of constitutive phosphorylation or dephosphorylation.
-
ChIP-seq analysis: Compare chromatin binding patterns of wild-type versus mutant CEBPA to identify genomic regions where binding is affected by Thr230 phosphorylation.
-
Transcriptomic analysis: Perform RNA-seq in cells expressing wild-type versus mutant CEBPA to identify genes whose expression is specifically regulated by Thr230 phosphorylation.
-
Protein interaction studies: Use co-immunoprecipitation or proximity ligation assays to identify proteins that preferentially interact with phosphorylated or non-phosphorylated CEBPA at Thr230.
-
Kinase inhibitor studies: Use inhibitors of candidate kinases to identify the upstream regulators of Thr230 phosphorylation.
How might CEBPA Thr230 phosphorylation contribute to acute myeloid leukemia pathogenesis?
CEBPA mutations are associated with acute myeloid leukemia (AML) . Researchers investigating the role of Thr230 phosphorylation in AML could:
-
Compare Thr230 phosphorylation levels between normal hematopoietic cells and AML samples.
-
Determine if CEBPA mutations found in AML affect Thr230 phosphorylation.
-
Investigate whether altered Thr230 phosphorylation correlates with specific AML subtypes or prognosis.
-
Study how leukemogenic factors affect the signaling pathways regulating Thr230 phosphorylation.
-
Examine whether targeting the kinases responsible for Thr230 phosphorylation affects AML cell growth and differentiation.
Technical Specifications of Phospho-CEBPA (Thr230) Antibody
Expected Molecular Weight of CEBPA Isoforms
| CEBPA Isoform | Molecular Weight (kDa) | Note |
|---|---|---|
| Full-length (p42) | 42 | Translated from AUG start codon |
| Extended (p45) | 45 | Translated from GUG start codon |
| Truncated (p30) | 30 | Alternative translation initiation |
Recommended Secondary Antibodies for Detection
| Secondary Antibody | Application | Dilution Range |
|---|---|---|
| HRP-Conjugated Anti-Rabbit IgG | Western Blot | 1:2000-1:5000 |
| AP-Conjugated Anti-Rabbit IgG | Western Blot, ELISA | 1:1000-1:3000 |
| Biotin-Conjugated Anti-Rabbit IgG | ELISA | 1:1000-1:5000 |
| FITC-Conjugated Anti-Rabbit IgG | IF | 1:100-1:500 |
