EIF4EBP1 (Ab-69) Antibody
Description
Introduction to EIF4EBP1 (Ab-69) Antibody
EIF4EBP1 (Ab-69) Antibody is a rabbit-derived polyclonal antibody specifically designed to detect endogenous levels of total 4E-BP1 protein in mammalian tissues and cell lines . The antibody targets a region around the threonine 70 phosphorylation site of 4E-BP1, a key regulatory site that influences 4E-BP1's activity and function in translation control . This specific antibody has been validated for multiple applications including Western blotting (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA), making it a versatile tool for researchers investigating translation regulation mechanisms and their dysregulation in various pathological conditions .
The importance of this antibody stems from the critical role that 4E-BP1 plays in regulating protein synthesis. As a substrate of the mechanistic target of rapamycin complex 1 (mTORC1), 4E-BP1 serves as a negative regulator of mRNA translation under normal conditions, but its function can be significantly altered in pathological states, particularly in cancer . Recent research has identified 4E-BP1 as potentially oncogenic in certain cancer contexts, contrary to its traditionally understood role as a translation suppressor .
Physical and Chemical Properties
The antibody is supplied as purified rabbit IgG in phosphate buffered saline (without Mg²⁺ and Ca²⁺), at pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol . This formulation ensures the stability and longevity of the antibody during storage and usage. The antibody has undergone affinity purification from rabbit antiserum through affinity chromatography using the epitope-specific immunogen, resulting in a highly purified antibody preparation .
Target Protein: Understanding 4E-BP1
To fully appreciate the utility of the EIF4EBP1 (Ab-69) Antibody, it is essential to understand its target protein, 4E-BP1, and its biological significance.
Structure and Function
4E-BP1 is encoded by the EIF4EBP1 gene (UniProt No. Q13541) and functions as a regulatory protein in the mRNA translation process . The protein is also known by several synonyms, including PHAS-I (Phosphorylated heat- and acid-stable protein regulated by insulin 1) . 4E-BP1 plays a critical role in cap-dependent translation by interacting with eIF4E, which is the cap-binding component of the eIF4F translation initiation complex .
In its hypophosphorylated state, 4E-BP1 binds to eIF4E, preventing the association of eIF4E with eIF4G and thereby inhibiting cap-dependent translation initiation . Upon phosphorylation by mTORC1 in response to various stimuli (such as nutrients, growth factors, or insulin), 4E-BP1 dissociates from eIF4E, allowing translation initiation to proceed .
Research Applications
The EIF4EBP1 (Ab-69) Antibody has proven valuable across multiple research applications, particularly in understanding the role of 4E-BP1 in normal cellular processes and disease states.
Western Blot Applications
The antibody has been validated for Western blot analysis with a recommended dilution range of 1:500-1:3000 . It effectively detects endogenous levels of total 4E-BP1 protein in cell lysates, as demonstrated in experiments with COS7 cells treated with epidermal growth factor (EGF) . Western blotting with this antibody allows researchers to monitor changes in 4E-BP1 expression levels as well as potential shifts in migration patterns that might indicate changes in phosphorylation status.
Immunohistochemistry Applications
For immunohistochemical analysis, the antibody is typically used at dilutions of 1:50-1:100 . It has been successfully employed in the analysis of paraffin-embedded human tissues, including colon carcinoma samples . This application enables researchers to visualize the expression and localization of 4E-BP1 in tissue sections, providing valuable insights into its role in tissue-specific contexts and pathological conditions.
ELISA Applications
The antibody is also suitable for enzyme-linked immunosorbent assay (ELISA) applications , allowing for quantitative assessment of 4E-BP1 levels in various sample types.
4E-BP1 in Cancer Research
The EIF4EBP1 (Ab-69) Antibody has become an important tool in cancer research, where the role of 4E-BP1 has increasingly gained attention.
Oncogenic Potential of 4E-BP1
Contrary to the traditional view of 4E-BP1 as a tumor suppressor through its inhibition of cap-dependent translation, recent research suggests that 4E-BP1 may function as an oncogene in certain contexts . Genome-scale shRNA screening has identified 4E-BP1 as a potential essential driver oncogene in many cancer cell lines, particularly in breast cancer models . This unexpected finding highlights the complex role of 4E-BP1 in cancer biology and underscores the importance of tools like the EIF4EBP1 (Ab-69) Antibody in elucidating these mechanisms.
Expression in Different Cancer Types
Studies using antibodies targeting 4E-BP1 have revealed that EIF4EBP1 is overexpressed in various cancer types:
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In breast cancer, 4EBP1 was found to be amplified and overexpressed in 8p11-amplified breast cancer cells, and its knockdown resulted in significant reductions in cell proliferation .
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In malignant gliomas, including isocitrate dehydrogenase (IDH)-wildtype glioblastomas, EIF4EBP1 mRNA is overexpressed relative to non-neoplastic brain tissue .
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In medulloblastoma, EIF4EBP1 mRNA expression is elevated in Group 3 tumors relative to Group 4, although levels were also high in the less aggressive WNT subgroup .
Transcriptional Regulation of EIF4EBP1
Research using specific antibodies has helped elucidate the mechanisms of EIF4EBP1 overexpression in cancer. In glioblastoma, for instance, EIF4EBP1 overexpression was found to result not from gene amplification or altered DNA methylation but from aberrant transcriptional activation by specific transcription factors, particularly MYBL2 and ETS1 . These findings highlight the multi-layered regulation of 4E-BP1 expression and function in cancer.
Practical Considerations for Antibody Usage
When working with the EIF4EBP1 (Ab-69) Antibody, several practical considerations should be taken into account to ensure optimal results.
Controls and Validation
For reliable results, appropriate positive and negative controls should be included in experimental designs. For blocking experiments, a synthetic peptide corresponding to the immunogen sequence can be used as a specificity control . The synthetic peptide used to generate the antibody was selected from the T69 region of human EIF4EBP1, and a 10 to 100-fold molar excess of blocking peptide relative to antibody is typically recommended .
Secondary Antibody Selection
For detection purposes, several secondary antibodies are compatible with this rabbit polyclonal primary antibody, including:
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Goat Anti-Rabbit IgG H&L Antibody (AP)
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Goat Anti-Rabbit IgG H&L Antibody (Biotin)
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Goat Anti-Rabbit IgG H&L Antibody (FITC)
The choice of secondary antibody depends on the specific detection method employed in each application.
Product Specs
Target Background
- BCH, an inhibitor of L-type amino acid transporter 1 (LAT1), reduces the phosphorylation of the mechanistic target of rapamycin kinase (mTOR) downstream target eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) in fibroblast-like synoviocytes from patients with rheumatoid arthritis. Silencing eIF4E neutralizes the stimulation of interleukin-17 on LAT1. PMID: 29198077
- Using an mTOR-specific signaling pathway phospho array, we observed that NVPBEZ235 significantly decreased phosphorylation of 4EBP1 (Thr70), a downstream target of mTORC1. PMID: 29845289
- High p-4E-BP1 expression was significantly associated with lymphovascular invasion (LVI) (p=0.003), perineural invasion (PNI) (p=0.001), tumor stage (p=0.024), nodal stage (p=0.000), metastatic status (p=0.027), and disease stage (p=0.001). PMID: 28242042
- Numerous protein kinases can be responsible for mTOR-independent 4E-BP1 phosphorylation in cancer. (Review) PMID: 28427795
- PI3K kinase activity is essential for maintaining 4E-BP1 stability. Our findings also suggest that 4E-BP1 plays a novel biological role in regulating cell cycle G2 checkpoint in response to IR stress, in association with controlling CHK2 phosphorylation. PMID: 28539821
- The findings suggest that mitotic CDK1-directed phosphorylation of delta-4E-BP1 may yield a gain of function, distinct from translation regulation, which could be significant in tumorigenesis and mitotic centrosome function. PMID: 27402756
- p4EBP1 was independently predictive for pathologic complete response in PIK3CA wild-type tumors. PMID: 26758558
- Data show that the 4EGI-1 compound induced apoptosis in nasopharyngeal carcinoma cells through the death receptor 5 (DR5) on 4E-BP1 dephosphorylation, exerting a positive influence on their anti-tumor activities. PMID: 26942880
- p4EBP1 overexpression was predominant in patients with metastasis to the regional lymph nodes in colorectal cancer. Moderate/high expression of p4EBP1 protein was significantly associated with adverse overall survival (OS) in patients. PMID: 28339030
- Rotterlin inhibits mTORC1 and 4EBP1 activity in melanoma cells, inhibiting protein synthesis and promoting cell death. PMID: 27343979
- p-4E-BP1 is more highly expressed in early gastric cancers than in advanced ones, and has limited potential as an independent prognostic biomarker in patients with gastric cancer. PMID: 25661069
- This study shows that the anticancer activity of perillyl alcohol is mediated via inhibition of 4E-BP1 signaling. PMID: 27394002
- 4EBP1 may serve as a funnel factor that converges the upstream proliferative oncogenic signals. PMID: 27026382
- Increased expression of miR-125a is associated with invasion and migration in ovarian cancer. PMID: 26646586
- 4E-BP1 was shown to be phosphorylated by other kinases besides mTOR, and overexpression of 4E-BP1 was found in different human carcinomas. (Review) PMID: 26901143
- Twist1 is correlated with p-4E-BP1 in predicting the prognostic outcome of NSCLC. PMID: 26360779
- Increased 4EBP1 abundance was a common feature in prostate cancer patients who had been treated with the PI3K pathway inhibitor BKM120; thus, 4EBP1 may be associated with drug resistance in human tumors. PMID: 26577921
- Results suggest that respiratory syncytial virus is a virus that still contains unknown mechanisms involved in the translation of their mRNAs through the alteration or modification of some translation factors, such as 4EBP1, possibly to favor its replicative process. PMID: 26305094
- eIF4E binding protein 1 expression has a role in clinical survival outcomes in colorectal cancer. PMID: 26204490
- It was concluded that over-activation of the mTORC1/4E-BP1/p21 pathway is a frequent and clinically relevant alteration in head and neck squamous cell carcinomas. PMID: 26832959
- The aim of the present work was to investigate the conformation of the intrinsically disordered protein 4E-BP1 in the native and partly folded states by limited proteolysis and to reveal regions with a high propensity to form an ordered structure. PMID: 24122746
- Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival. PMID: 25999352
- Results suggest that blocking both the mTOR kinase downstream targets 4E-BP1 protein and p70 S6 kinase 1, but not p70 S6 kinase 1 alone, prevents the migration of retinal pigment epithelium (RPE) cells. PMID: 26427479
- Phosphorylation site affected the prognostic significance of 4EBP1 in non-small cell lung cancer. PMID: 26097581
- 4EBP1 is not completely unstructured, but contains a pre-structured helix. PMID: 25431930
- Inactivation of 4E-BP1 using Ku-0063794 may be a promising novel approach for muscle-invasive bladder cancer. PMID: 25618298
- mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via an Akt-independent mechanism. PMID: 25762619
- Mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. PMID: 25883264
- In colorectal carcinoma, total expression levels of 4E-BP1 increased only in the premalignant state of the disease and decreased (but highly phosphorylated or inactivated) or abolished upon malignancy. PMID: 25755728
- Results show that high expression of p70S6K and 4EBP1 proteins may act as valuable independent biomarkers to predict poor prognosis of nasopharyngeal carcinoma patients. PMID: 25165983
- The C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and it partially overlaps with the binding site of 4EGI-1. PMID: 26170285
- Effect of temperature on the conformation of natively unfolded protein 4E-BP1 in aqueous and mixed solutions containing trifluoroethanol and hexafluoroisopropanol. PMID: 25503819
- ShcA drives breast tumor growth and angiogenesis in vivo in a 4E-BP-dependent manner. PMID: 24837366
- Studied conditions that increase the sensitivity of cancer cells to MK-2206. We found a reduction by salinomycin of Akt and downregulation of pAkt, pGSk3beta, pTSC2, and p4EBP1 by cotreatment with MK-2206. PMID: 25114899
- Tanshinone IIA inhibits HIF-1alpha and VEGF expression in breast cancer cells via the mTOR/p70S6K/RPS6/4E-BP1 signaling pathway. PMID: 25659153
- Certain Akt/mTOR/4E-BP1 pathway signals could be novel therapeutic targets for Merkel cell carcinomas regardless of Merkel cell polyomavirus infection status. PMID: 25466966
- 4E-BP1 is a trigger for parthenolide-induced autophagy. PMID: 25482447
- Acquired drug resistance to antineoplastic agents is regulated in part by 4E-BP1. PMID: 24354477
- Results show that loss-of-function of TBC1D7 protein was associated with an increase in the phosphorylation of 4EBP1, a direct downstream target of mTORC1. PMID: 24515783
- Overexpression of phosphorylated 4E-binding protein 1 is associated with lymph node metastasis in hilar cholangiocarcinoma. PMID: 24706262
- The tumor marker eRF3B can change the cell cycle and influence the phosphorylation status of 4E-BP1. PMID: 24466059
- The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer. PMID: 24131622
- p-4E-BP1 may have a role in response to the mTOR inhibitors and progression-free survival. PMID: 24307346
- mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria-related mRNAs via inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). PMID: 24206664
- Rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1; there is a molecular cross-talk between the mTORC1 and MNK1 pathways. PMID: 24401275
- Overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote Coxsackievirus B3-induced apoptosis. PMID: 24030155
- Our results suggest that long-term repeated viral delivery of 4E-BP1 may provide a useful tool for designing lung cancer treatment. PMID: 23640516
- The results indicate mTOR-independent phosphorylation of S6K1 and 4E-BP1 and suggest MEK/ERK/RSK1-dependent phosphorylation of eIF4B during skeletal muscle contraction. PMID: 23707523
- The study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1. PMID: 23814053
- Data therefore suggest that HIF-1alpha contributes to 4E-BP1 gene expression under different conditions. PMID: 23175522
