EIF4EBP1 Antibody

What is EIF4EBP1 and what signaling pathways regulate its function?

EIF4EBP1 (also known as 4E-BP1) is a negative regulator of mRNA translation that functions by binding to eIF4E, preventing its assembly into the EIF4F complex and inhibiting cap-dependent translation. 4E-BP1 mediates the regulation of protein translation by growth factors, hormones, and other stimuli that signal through the PI3 kinase and mTORC1 pathways .

When unphosphorylated, 4E-BP1 binds tightly to eIF4E, blocking cap-dependent translation. Upon phosphorylation by mTOR, 4E-BP1 releases eIF4E, allowing translation initiation to proceed. This phosphorylation occurs in a hierarchical manner, with Thr37/46 phosphorylation priming 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 .

What are the optimal validation methods for EIF4EBP1 antibodies?

Proper validation of EIF4EBP1 antibodies should include multiple complementary approaches:

• Western blot analysis: Verify specificity using both positive controls (cell lines known to express 4E-BP1 such as HeLa, C2C12, NIH/3T3) and negative controls (4E-BP1 knockout cell lines) .

• Peptide competition assays: Demonstrate that antibody binding can be blocked by synthetic immunogen peptides .

• Phosphorylation-state specificity: For phospho-specific antibodies, compare untreated versus treated conditions (e.g., serum-starved versus serum-stimulated cells) and include λ-phosphatase-treated samples as negative controls .

• Cross-reactivity assessment: Test reactivity across species (human, mouse, rat) if planning cross-species experiments .

What techniques can be used to study EIF4EBP1 and what antibody applications are most common?

Multiple techniques can be employed to study EIF4EBP1, each requiring specific antibody characteristics:

Researchers should select antibodies validated for their specific application and consider using multiple antibodies targeting different epitopes to confirm results .

What are the optimal conditions for immunohistochemical detection of EIF4EBP1?

For optimal immunohistochemical detection of 4E-BP1, the following protocol has been successfully employed:

• Tissue preparation: Deparaffinize sections and perform antigen retrieval using citrate buffer at 98°C for 20 minutes, then cool to room temperature.

• Blocking: Block with 2% horse serum, followed by avidin and biotin blocking solutions (10 minutes each).

• Primary antibody: Incubate with anti-4E-BP1 antibody (e.g., monoclonal rabbit anti-4E-BP1, 1:200 dilution) for 2 hours at 37°C.

• Detection: Apply an appropriate detection system such as Dako REAL detection system with alkaline phosphatase/RED.

• Counterstaining: Counterstain with hematoxylin solution according to Mayer.

• Evaluation: Score using the Immune Reactive Score (IRS) system as detailed below .

For tissue microarrays, taking three representative cores (each 1mm in diameter) from blocks exhibiting at least 80% viable tumor tissue is recommended .

How should the Immune Reactive Score (IRS) for EIF4EBP1 staining be calculated and interpreted?

The Immune Reactive Score (IRS) provides a semi-quantitative assessment of 4E-BP1 expression:

Step 1: Score the percentage of positive cells:

• Grade 0 = 0-19%

• Grade 1 = 20-39%

• Grade 2 = 40-59%

• Grade 3 = 60-79%

• Grade 4 = 80-100%

Step 2: Evaluate staining intensity:

• Grade 0 = None

• Grade 1 = Low

• Grade 2 = Moderate

• Grade 3 = Strong

Step 3: Calculate the IRS by multiplying the percentage grade by the intensity grade (range: 0-12).

Interpretation:

• IRS 0-6 = "Low" expression level

• IRS 7-12 = "High" expression level

This scoring system has been used in prognostic studies of neuroblastoma and other cancers to stratify patients based on 4E-BP1 expression levels .

What sample preparation protocols yield optimal results for analyzing EIF4EBP1 phosphorylation by Western blot?

For optimal Western blot analysis of 4E-BP1 phosphorylation states:

• Cell treatment: Compare experimental conditions that alter 4E-BP1 phosphorylation (e.g., serum starvation followed by stimulation with 20% FBS, which increases phosphorylation).

• Lysis conditions: Use buffers that preserve phosphorylation states, containing phosphatase inhibitors.

• Gel selection: Use 12-15% gels for optimal resolution of the different phosphorylation states, as 4E-BP1 migrates between 15-20 kDa.

• Antibody selection:

  • For total 4E-BP1: Use antibodies that recognize all forms regardless of phosphorylation state.

  • For specific phosphorylation sites: Use antibodies specific for phospho-Thr37/46, phospho-Ser65, or phospho-Thr70.

• Controls:

  • Phosphatase treatment of lysates to demonstrate specificity of phospho-antibodies.

  • Comparison between stimulated and unstimulated conditions.

  • Loading controls such as GAPDH.

• Analysis: The phosphorylation status of 4E-BP1 can be visualized as a ladder of bands with different electrophoretic mobilities, with the most phosphorylated forms migrating more slowly .

EIF4EBP1 in Cancer Research

EIF4EBP1 transcription is regulated by multiple transcription factors in a context-dependent manner:

• MYCN in neuroblastoma: MYCN directly binds to the EIF4EBP1 promoter at three distinct E-boxes, upregulating its transcriptional activity .

• MYC in other cancers: MYC directly controls EIF4EBP1 transcription in colon adenocarcinoma and prostate cancer cells, with MYC expression strongly correlating with EIF4EBP1 expression, particularly in Group 3 medulloblastoma .

• Other transcription factors:

  • Androgen receptor in prostate cancer

  • ETS1 and MYBL2 in glioblastoma, identified through chromatin immunoprecipitation (ChIP) and functional validation

  • HIF-1A and E2F6 also bind to the EIF4EBP1 promoter region

Notably, EIF4EBP1 overexpression in malignant gliomas is not due to gene amplification or altered DNA methylation but results from aberrant transcriptional activation .

How should researchers interpret contradictory findings about EIF4EBP1's role as a tumor suppressor versus oncogene?

The paradoxical roles of EIF4EBP1 as both tumor suppressor and oncogene require careful experimental design and interpretation:

• Context-dependent function: 4E-BP1 can exert tumor suppressive functions in some contexts (e.g., head and neck squamous cell carcinoma mouse models) while promoting tumor growth in others (e.g., breast cancer models through angiogenesis facilitation) .

• Phosphorylation status: The ratio of phosphorylated to unphosphorylated 4E-BP1 may be more important than total expression levels. Active (unphosphorylated) 4E-BP1 inhibits translation, whereas inactive (phosphorylated) 4E-BP1 allows translation to proceed .

• Experimental approach recommendations:

  • Analyze both total 4E-BP1 and phosphorylated forms in the same samples

  • Correlate with upstream regulators (e.g., mTOR activity)

  • Assess functional outcomes of modulating 4E-BP1 activity

  • Consider genetic background and molecular subtype

• Reconciling contradictions: In neuroblastoma, contradictory reports showed EIF4EBP1 upregulated in MYCN-amplified tumors in some studies, while others reported higher levels in favorable stages compared to advanced stage 4 tumors. These contradictions were resolved through comprehensive analysis of multiple cohorts, confirming that high EIF4EBP1 expression associates with poor outcomes .

What phosphorylation sites of EIF4EBP1 are most critical for research applications and how are they regulated?

The phosphorylation of 4E-BP1 occurs in a hierarchical manner with specific sites serving as key research targets:

• Thr37/46:

  • Primary phosphorylation sites by mTOR

  • Serve as priming sites for subsequent phosphorylation

  • Do not prevent binding to eIF4E on their own

  • Detectable with specific phospho-Thr37/46 antibodies

• Ser65:

  • Subsequent phosphorylation site

  • Important for dissociation of 4E-BP1 from eIF4E

  • Phosphorylation can be detected in serum-stimulated cells

• Thr70:

  • Later phosphorylation site

  • Works with Ser65 to release eIF4E

  • Can be rapidly induced by serum stimulation

Research applications typically involve:

• Monitoring changes in phosphorylation at these sites in response to treatments

• Comparing phosphorylation patterns across different disease states

• Evaluating the efficacy of mTOR inhibitors

Phosphorylation analysis by Western blot reveals a ladder of bands representing different phosphorylation states, with the most heavily phosphorylated forms migrating slower on SDS-PAGE .

What methods are most effective for studying the interaction between EIF4EBP1 and transcription factors that regulate its expression?

Several complementary approaches have proven effective for studying transcriptional regulation of EIF4EBP1:

• Chromatin Immunoprecipitation (ChIP):

  • Identifies direct binding of transcription factors to the EIF4EBP1 promoter and regulatory regions

  • ChIP-sequencing data has demonstrated binding of FOXM1, ETS1, E2F1, E2F6, MYBL2, and HIF-1A to the EIF4EBP1 promoter region, exon 1, and intron 1

• Luciferase Reporter Assays:

  • Measure the ability of candidate transcription factors to activate the EIF4EBP1 promoter

  • Successfully employed to validate MYBL2, ETS1, HIF-1A, and E2F6 as potential EIF4EBP1 regulators

• Genetic Manipulation:

  • Knockdown or overexpression of candidate transcription factors followed by assessment of EIF4EBP1 expression

  • Transient knockdown experiments identified MYBL2 and ETS1 as relevant transcriptional drivers of EIF4EBP1 expression in malignant glioma cells

• Correlation Analysis in Patient Samples:

  • Analysis of co-expression patterns between EIF4EBP1 and potential regulators

  • Significant co-expression between MYCN and EIF4EBP1 in neuroblastoma helped identify this regulatory relationship

The regulatory region of EIF4EBP1 extends beyond the promoter to include exon 1 and the 5' region of intron 1, as indicated by histone modification patterns (H3K27 acetylation and H3K4 trimethylation) .

How can EIF4EBP1 antibodies be used in combination with other techniques to comprehensively analyze its function?

Comprehensive analysis of EIF4EBP1 function requires integrating multiple techniques:

• Multi-antibody approach:

  • Use antibodies against total 4E-BP1 alongside phospho-specific antibodies

  • Combine with antibodies against upstream regulators (mTOR, PI3K) and downstream effectors (eIF4E, translation products)

  • Example: Western blot analysis with phospho-4E-BP1 (Thr37/46) antibody alongside total 4E-BP1 antibody allows assessment of the proportion of phosphorylated protein

• Transcriptional-translational integration:

  • RNA-sequencing to assess EIF4EBP1 mRNA levels

  • Protein analysis with EIF4EBP1 antibodies

  • Polysome profiling to evaluate translation efficiency

  • Example: In medulloblastoma studies, researchers analyzed EIF4EBP1 mRNA expression in publicly available data sets and correlated with MYC expression

• Functional validation techniques:

  • CRISPR/Cas9 knockout of EIF4EBP1 followed by antibody validation

  • Proximity ligation assays to detect protein-protein interactions in situ

  • Drug inhibition studies targeting upstream regulators

  • Example: Anti-BUB1 + EIF4EBP1 antibody pairs can be used for proximity ligation assays to detect protein-protein interactions

• Clinical correlation approaches:

  • Tissue microarrays with IHC staining for 4E-BP1

  • Correlation with patient outcome data

  • Example: IHC analysis of 4E-BP1 in neuroblastoma tissues confirmed mRNA-based associations and showed that high 4E-BP1 protein expression associates with unfavorable histology

What are common technical challenges when working with EIF4EBP1 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with EIF4EBP1 antibodies:

• Multiple band detection in Western blots:

  • Challenge: 4E-BP1 appears as multiple bands (15-20 kDa) due to different phosphorylation states.

  • Solution: Use phospho-specific antibodies to identify specific modified forms; include phosphatase-treated controls to collapse bands to unphosphorylated form .

• Cross-reactivity with related proteins:

  • Challenge: Potential cross-reactivity with other 4E-BP family members (4E-BP2, 4E-BP3).

  • Solution: Validate antibody specificity using 4E-BP1 knockout cells; perform peptide competition assays .

• Variable immunohistochemistry staining:

  • Challenge: Inconsistent staining patterns or background.

  • Solution: Optimize antigen retrieval (citrate buffer at 98°C for 20 min); use standardized IRS scoring system; include multiple tissue cores per sample in TMAs .

• Reconciling protein and mRNA data:

  • Challenge: Discrepancies between protein and mRNA expression levels.

  • Solution: Analyze both in parallel; consider post-transcriptional regulation and protein stability .

• Detecting subtle phosphorylation changes:

  • Challenge: Small changes in phosphorylation may be functionally significant but difficult to detect.

  • Solution: Use Phos-tag gels for enhanced separation of phosphorylated species; quantify band intensities with densitometry and normalize to total protein .

What quality control methods should be implemented when using EIF4EBP1 antibodies for quantitative analysis?

For reliable quantitative analysis using EIF4EBP1 antibodies, implement these quality control measures:

• Antibody validation:

  • Verify specificity using Western blot against multiple cell lines

  • Confirm specific detection of a single protein band that can be blocked by immunogen peptide

  • Test reactivity across relevant species if conducting comparative studies

• Standardization protocols:

  • For immunohistochemistry: Use standardized scoring systems like IRS (0-12) or H-score (0-300)

  • For Western blot: Include calibration samples on each gel for inter-gel normalization

  • For ELISA: Run standard curves on each plate

• Appropriate controls:

  • Positive controls: Well-characterized cell lines (HeLa, C2C12, NIH/3T3, 293 cells)

  • Negative controls: 4E-BP1 knockout cell lines, secondary antibody only, isotype controls for flow cytometry

  • Treatment controls: Compare untreated versus treated conditions (e.g., serum-starved versus serum-stimulated)

• Technical replicates:

  • Run samples in triplicate when possible

  • For tissue analysis, use multiple cores per sample in tissue microarrays

  • Report variability metrics (standard deviation, coefficient of variation)

• Independent validation:

  • Use multiple antibodies targeting different epitopes

  • Employ orthogonal techniques to confirm findings (e.g., mass spectrometry)

  • Validate findings across independent patient cohorts