Phospho-FOXO4 (T451) Antibody

What is FOXO4 and why is T451 phosphorylation significant?

FOXO4 (Forkhead box protein O4) is a transcription factor belonging to the FOXO class that regulates cell cycle progression and cell death. It is negatively regulated by PKB/c-Akt in response to insulin/IGF signaling . The phosphorylation at threonine 451 (T451) represents a critical post-translational modification that occurs independently of PKB activation and significantly impacts FOXO4's transcriptional activity .

T451 phosphorylation is particularly significant because it occurs in response to oxidative stress, notably through hydrogen peroxide (H₂O₂) exposure, and plays a crucial role in activating FOXO4 . This activation mechanism represents a distinct regulatory pathway from the classic insulin-mediated inhibition of FOXO transcription factors.

How does the FOXO4 phosphorylation at T451 differ from other phosphorylation sites?

T451 phosphorylation differs from other FOXO4 modifications in several important ways:

• Unlike PKB/Akt-mediated phosphorylation sites that inhibit FOXO4, T451 phosphorylation activates FOXO4 transcriptional activity

• T451 phosphorylation is specifically induced by oxidative stress conditions and not by insulin signaling

• The phosphorylation pathway involves the small GTPase Ral and JNK kinases rather than the insulin/PI3K/Akt pathway

• T451 phosphorylation leads to FOXO4 nuclear translocation (opposite effect to Akt-mediated phosphorylation)

Notably, experiments show that mutation of T451 to alanine (preventing phosphorylation) almost completely blocks transcriptional activity, while phospho-mimicking mutations (T451E) enhance transcriptional activity compared to wild-type FOXO4 .

What are the validated applications for Phospho-FOXO4 (T451) antibodies?

Phospho-FOXO4 (T451) antibodies have been validated for multiple experimental applications:

The antibodies have demonstrated reactivity with both human and mouse samples, though researchers should note that T451 is not conserved between human and mouse FOXO4, which may affect experimental design when working with mouse models .

How should I design controls when using Phospho-FOXO4 (T451) antibody?

Proper experimental controls are critical for interpreting results with phospho-specific antibodies:

• Positive controls:

  • Cells treated with H₂O₂ (20-200 μM) to induce T451 phosphorylation

  • Cells expressing constitutively active Ral or RalGEFs, which enhance T451 phosphorylation

• Negative controls:

  • Untreated cells (baseline phosphorylation levels)

  • Cells expressing FOXO4-T451A mutant (cannot be phosphorylated at this site)

  • Cells treated with JNK inhibitors prior to oxidative stress induction

  • Cells expressing dominant-negative Ral (RalN28), which blocks T451 phosphorylation

• Specificity controls:

  • Parallel detection with total FOXO4 antibody to normalize phosphorylation levels

  • Phosphatase treatment of samples to demonstrate phospho-specificity

Research has confirmed that the T451P antibody does not recognize FOXO4-T451A mutants isolated from either untreated or H₂O₂-treated cells, validating its specificity .

What are the key buffer and storage considerations for Phospho-FOXO4 (T451) antibody?

Optimal handling of phospho-specific antibodies is essential for maintaining their specificity and sensitivity:

When preparing to use the antibody, allow it to equilibrate to room temperature before opening the vial to prevent condensation that could introduce contaminants .

How can I optimize signal detection when using Phospho-FOXO4 (T451) antibody?

Optimizing signal detection requires careful consideration of several experimental parameters:

• Sample preparation:

  • Rapid sample collection and processing is critical as phosphorylation states can change quickly

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status

  • For H₂O₂-induced phosphorylation, optimal treatment is approximately 20 μM for detecting initial phosphorylation events

• Antibody concentration optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Western blot starting dilution: 1/500 to 1/2000

  • ELISA starting dilution: 1/40000

• Signal amplification strategies:

  • Enhanced chemiluminescence detection for Western blots

  • For low abundance proteins, consider using signal amplification systems

  • Adjust exposure times to avoid signal saturation while maximizing detection

• Background reduction:

  • Extend blocking time to reduce non-specific binding

  • Include detergents like Tween-20 in wash buffers

  • Consider using more sensitive detection methods for low abundance targets

What is the relationship between oxidative stress, Ral, JNK, and FOXO4 T451 phosphorylation?

The pathway connecting oxidative stress to FOXO4 activation involves a signaling cascade with several key components:

• Oxidative stress induction: H₂O₂ treatment (even at low concentrations of 20 μM) or tumor necrosis factor α (TNFα) exposure generates cellular reactive oxygen species

• Ral activation: Oxidative stress triggers rapid activation of the small GTPase Ral, observable through increased Ral-GTP levels in pull-down assays

• JNK activation: Active Ral leads to phosphorylation and activation of JNK (c-Jun N-terminal kinase)

• FOXO4 phosphorylation: Activated JNK phosphorylates FOXO4 at T447 and T451 residues

• FOXO4 activation: Phosphorylation at these sites increases FOXO4 transcriptional activity

This pathway has been confirmed through multiple experimental approaches including dominant-negative Ral expression (RalN28), which completely blocks T451 phosphorylation, and JNK kinase inhibitor studies, which prevent T451 phosphorylation following oxidative stress .

How can I investigate the functional consequences of T451 phosphorylation in cellular models?

To assess the functional impact of FOXO4 T451 phosphorylation, consider these methodological approaches:

• Transcriptional activity assays:

  • Luciferase reporter assays using FOXO-responsive elements

  • Compare wild-type FOXO4 with phospho-deficient (T451A) and phospho-mimetic (T451E) mutants

  • Include RalN28 co-expression to block the endogenous phosphorylation pathway

• Cellular localization studies:

  • Immunofluorescence or fractionation experiments to track FOXO4 nuclear translocation after H₂O₂ treatment

  • GFP-tagged FOXO4 variants for live-cell imaging

• Target gene expression analysis:

  • qRT-PCR or RNA-seq to measure changes in FOXO4-regulated genes

  • ChIP assays to assess binding to target promoters

• Functional outcome measurements:

  • Glucose deprivation experiments to assess cellular protection

  • Studies have shown that inhibition of Ral signaling and expression of FOXO4-T447/451A mutant reduced the ability of FOXO4 to protect cells from glucose deprivation, while the phospho-mimicking T447/451D mutant enhanced protection

• JNK dependency testing:

  • Use JNK1,2−/− cells to confirm pathway dependence

  • Reintroduction of JNK in knockout cells restores FOXO4 transcriptional activity

What could cause false negative results when using Phospho-FOXO4 (T451) antibody?

Several technical issues may lead to false negative results when attempting to detect phosphorylated FOXO4:

• Sample handling issues:

  • Rapid dephosphorylation during sample preparation (insufficient phosphatase inhibitors)

  • Inadequate cell lysis leading to incomplete protein extraction

  • Protein degradation during extended processing

• Stimulation conditions:

  • Suboptimal H₂O₂ concentration (20 μM is typically effective)

  • Incorrect timing of stimulation (phosphorylation is time-dependent)

  • Cell type variations in response to oxidative stress

• Detection challenges:

  • Antibody quality issues or inappropriate dilution

  • Insufficient exposure time in Western blots

  • Blocking buffer interference with epitope recognition

• Biological variations:

  • Low endogenous expression of FOXO4

  • Species differences (T451 is not conserved between human and mouse FOXO4)

  • Concurrent activation of phosphatases that rapidly dephosphorylate T451

How do I interpret contradictory results between phospho-T451 and other FOXO4 phosphorylation sites?

Contradictory phosphorylation patterns may reflect complex regulatory mechanisms:

• Pathway crosstalk: Oxidative stress and insulin signaling may have opposing effects on different FOXO4 phosphorylation sites. High H₂O₂ concentrations (200 μM) can activate both JNK-mediated T451 phosphorylation and PKB/Akt pathways that phosphorylate inhibitory sites .

• Temporal dynamics: Different phosphorylation events may occur with distinct kinetics. T451 phosphorylation may be rapid and transient, while other modifications occur with different timing.

• Subcellular compartmentalization: Phosphorylation at T451 promotes nuclear localization, while Akt-mediated phosphorylation promotes cytoplasmic retention. The cellular fraction analyzed may influence results.

• Signal strength effects: Low levels of oxidative stress (20 μM H₂O₂) induce T451 phosphorylation without activating PKB, while higher concentrations (200 μM) activate both pathways .

• Methodological considerations: Phospho-specific antibodies for different sites may have varying sensitivities and specificities, complicating direct comparisons.

How can phospho-FOXO4 (T451) be utilized to study oxidative stress responses?

The phospho-FOXO4 (T451) antibody serves as a valuable tool for investigating cellular responses to oxidative stress:

• Oxidative stress biomarker: Detection of T451 phosphorylation serves as a sensitive biomarker for low-level oxidative stress, detectable at H₂O₂ concentrations as low as 20 μM

• Homeostatic feedback loops: FOXO4 activation via T451 phosphorylation participates in a negative feedback loop to control cellular ROS levels, offering insights into cellular homeostasis mechanisms

• ROS-inducer screening: The antibody can be used to screen compounds for their ability to induce ROS production by monitoring T451 phosphorylation

• Redox-sensitive signaling: Studies of T451 phosphorylation timing and intensity can reveal new insights into how redox signals are processed intracellularly

• TNFα signaling pathway: Research confirms that TNFα, a ligand known to increase cellular H₂O₂ levels, activates FOXO4 transcriptional activity through cellular ROS, Ral, and JNK, with dosage-dependent effects measurable using phospho-T451 antibodies

What are the considerations for multiplexing phospho-FOXO4 (T451) with other phospho-specific antibodies?

When designing multiplexed phospho-protein detection experiments:

• Antibody compatibility:

  • Ensure primary antibodies are raised in different host species

  • Consider using sequential detection methods to avoid cross-reactivity

  • Phospho-FOXO4 (T451) antibodies are typically raised in rabbit

• Phosphorylation dynamics:

  • Plan for differential timing of phosphorylation events

  • Consider time-course experiments to capture transient modifications

• Signal separation strategies:

  • Use differently labeled secondary antibodies

  • For Western blots, strip and reprobe or use spectrally separated fluorophores

  • For fluorescence microscopy, select fluorophores with minimal spectral overlap

• Controls for multiplexed detection:

  • Single antibody controls to establish baseline signals

  • Phosphatase-treated controls to confirm specificity

  • Stimulated and unstimulated samples to verify responsiveness

• Normalization approaches:

  • Include antibodies against total FOXO4 and other proteins of interest

  • Use housekeeping proteins as loading controls

  • Consider internal normalization controls for phospho/total ratios

By following these guidelines, researchers can design robust experiments to investigate the complex interplay between FOXO4 T451 phosphorylation and other signaling events in response to oxidative stress and related cellular challenges.