FOXO3 (Ab-253) Antibody

What is the biological significance of FOXO3a phosphorylation at Ser253?

Phosphorylation of FOXO3a at Ser253 is a critical regulatory mechanism that determines its subcellular localization and function. When FOXO3a is phosphorylated at Ser253 (primarily by Akt/PKB), it associates with 14-3-3 proteins, which retain it in the cytoplasm and prevent its transcriptional activity. This phosphorylation event is part of the PI3K/Akt signaling pathway activated by growth factors and insulin.

In the absence of survival factors or under oxidative stress, FOXO3a becomes dephosphorylated, translocates to the nucleus, and triggers various cellular responses including apoptosis via a Fas ligand-dependent mechanism. The S253 site is particularly crucial as it regulates the nuclear/cytoplasmic shuttling of FOXO3a .

Which experimental applications are validated for phospho-FOXO3a (Ser253) antibodies?

Based on the available data, phospho-FOXO3a (Ser253) antibodies have been validated for multiple applications:

The antibody has been tested on various samples including human uterus cancer tissue (IHC) and cell lines such as MCF-7 treated with IGF and NIH/3T3 treated with Calyculin A .

What molecular weight should be expected when detecting phospho-FOXO3a (Ser253) by Western blot?

While the calculated molecular weight of FOXO3a is approximately 71 kDa, the observed molecular weight on Western blots is typically around 97 kDa. This discrepancy is common and can be attributed to several factors:

• Post-translational modifications including phosphorylation

• Protein structure and charge affecting mobility

• Different modified forms present simultaneously

For example, Elabscience's phospho-FOXO3a (Ser253) antibody datasheet specifically notes: "The actual band is not consistent with the expectation. Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

How can researchers effectively validate the specificity of phospho-FOXO3a (Ser253) antibodies?

Multiple approaches should be employed to validate antibody specificity:

• Phosphatase treatment controls: Treat one sample with lambda phosphatase to remove phosphorylation and confirm signal loss.

• Positive controls: Use cell lines with known FOXO3a phosphorylation status:

  • MCF-7 cells treated with IGF

  • NIH/3T3 cells treated with Calyculin A (100 nM) for 30 minutes after serum starvation

• Stimulation/inhibition experiments:

  • Stimulate the PI3K/Akt pathway with growth factors

  • Inhibit the pathway with PI3K inhibitor LY294002 (10 μM) or PKB inhibitor VIII (10 μM)

• siRNA knockdown: Confirm signal reduction using validated FOXO3a siRNAs as demonstrated in several studies .

• Blocking peptide: Use a phospho-Ser253 specific peptide to competitively inhibit antibody binding .

• Cross-validation: Compare results from multiple antibodies targeting the same epitope from different vendors.

What are the critical parameters for optimal detection of phospho-FOXO3a (Ser253) in Western blotting?

Based on published protocols and technical data, the following parameters are critical:

Sample preparation:

• Fresh lysates yield better results

• Include phosphatase inhibitors (e.g., Calyculin A, sodium orthovanadate)

• Maintain cold temperatures during lysis to preserve phosphorylation

Western blot conditions:

• Antibody dilution: 1:500-1:2000 based on vendor recommendations

• Incubation time: 1 hour at room temperature or overnight at 4°C

• Detection method: ECL Basic Kit for visualization

Buffer composition:

• Blocking buffer: 3% nonfat dry milk in TBST (some phospho-antibodies work better with BSA)

• Loading: 25 μg protein per lane is typically sufficient

Positive control treatment:

• Serum starvation overnight followed by treatment with Calyculin A (100 nM) at 37°C for 30 minutes

How does the FOXO3a-NF-κB RelA interaction influence experimental design when studying myeloid cell function?

The discovery that FOXO3 forms protein complexes with NF-κB RelA has significant implications for experimental design when studying myeloid cells:

• Complex formation considerations:

  • FOXO3 binds NF-κB RelA in the cytosol

  • This interaction prevents FOXO3 degradation

  • It also prevents NF-κB RelA nuclear translocation

  • The interaction occurs near the FOXO3 transactivation domain

• Experimental approaches to study this interaction:

  • Co-immunoprecipitation with anti-FOXO3 or anti-NF-κB RelA antibodies

  • Cell fractionation to analyze cytoplasmic versus nuclear distribution

  • Deletion mutants to map interaction domains (specific deletion in FOXO3 containing the DNA binding domain restores NF-κB RelA activation)

• Functional implications to consider:

  • In tumor-associated dendritic cells, this interaction affects immune-suppressive properties

  • When designing experiments targeting either protein, the mutual regulation must be considered

  • Treatment with IL-6 (20 ng/ml) can be used to modulate this interaction

What factors contribute to variability in phospho-FOXO3a (Ser253) signal intensity across different experimental conditions?

Several factors can contribute to signal variability:

• Phosphorylation dynamics:

  • Rapid dephosphorylation occurs during sample processing

  • Cycloheximide studies show phosphorylated FOXO3a can be rapidly degraded (within 5-30 minutes)

• Cell type differences:

  • Expression levels of FOXO3a vary across tissues

  • The PI3K/Akt pathway activation status differs between cell types

  • Different phosphatases (e.g., PP2A) may be differentially expressed

• Sample handling:

  • Time between collection and fixation/lysis affects phosphorylation

  • Temperature fluctuations during processing

• Antibody-specific factors:

  • Lot-to-lot variation in antibody performance

  • Some lots contain BSA which may interfere with certain applications

• Technical variability:

  • Incomplete transfer during Western blotting

  • Inconsistent blocking or washing steps

What methodological approaches can distinguish between nuclear and cytoplasmic phospho-FOXO3a (Ser253)?

To accurately assess FOXO3a subcellular localization:

• Cell fractionation protocol:

  • Separate nuclear and cytoplasmic fractions using established protocols

  • Include markers for nuclear (LAMIN-B1) and cytoplasmic (β-ACTIN) fractions as controls

  • Western blot both fractions for phospho-FOXO3a (Ser253)

• Immunofluorescence co-localization:

  • Perform IF with phospho-FOXO3a (Ser253) antibody (1:200-1:1000 dilution)

  • Co-stain with nuclear marker (DAPI)

  • Use confocal microscopy for precise localization

• Chromatin immunoprecipitation (ChIP):

  • For nuclear active FOXO3a, perform ChIP with total FOXO3a antibody

  • Target known FOXO3a binding sites such as the androgen receptor promoter

  • Primers: Forward 5′-TCTCCCTTCTGCTTGTCCTGGT-3′, Reverse 5′-TAGGCTCCAAAGCAGAAGCGAT-3′

• Analytical considerations:

  • Phospho-Ser253 FOXO3a is predominantly cytoplasmic

  • Cellular localization is described as "Cytoplasm>cytosol. Nucleus. Translocates to the nucleus upon oxidative stress and in the absence of survival factors."

What approaches can resolve contradictory findings when studying FOXO3a function using phospho-specific antibodies?

When faced with contradictory results:

• Validate phosphorylation status with multiple techniques:

  • Use both phospho-specific and total FOXO3a antibodies

  • Employ mass spectrometry to confirm phosphorylation sites

  • Use Phos-tag gels to separate phosphorylated from non-phosphorylated forms

• Consider context-dependent functions:

  • FOXO3a exhibits seemingly contradictory functions (promotes both apoptosis and stress resistance)

  • Context matters: "In contrast to FoxO3a's better known functions of inhibiting cell proliferation and promoting apoptosis, FoxO3a also participates in protecting cells when exposed to unfavorable conditions."

  • Reactive oxygen species (ROS) are linked to the activation of FOXO3a in stress protection

• Examine kinase-phosphatase balance:

  • "The imbalance between kinases and phosphatase(s) can greatly affect a cell's fate by curbing FoxO3a function"

  • PP2A dephosphorylates T32/S253 residues and inhibits 14-3-3 binding to FOXO3a

• Control for bimodal regulation:

  • "AKT overexpression increases the steady-state levels of FoxO3 protein in a manner dependent on AKT activity and its ability to bind FoxO3"

  • This suggests more complex modes of regulation indicating positive roles in addition to reported negative roles

How can enhancer mapping techniques be integrated with FOXO3a phosphorylation studies?

Recent research has revealed FOXO3's role in enhancer activation, suggesting integrated approaches:

• Enhancer identification methodology:

  • ChIP-seq for FOXO3a binding sites

  • Histone modification profiling: "The canonical chromatin signature of distant enhancers is the presence of Histone H3 Lysine 4 monomethylation (H3K4me1), while trimethylation (H3K4me3) is generally absent"

  • Active enhancers are marked by H3K27 acetylation

• Experimental validation of enhancers:

  • ChIP-qPCR with antibodies recognizing H3K4me1, H3K4me3, and H3K27Ac

  • Luciferase reporter assays: "Four out of five sequences were responsive to FOXO3 induction in a luciferase-reporter assay"

  • Test orientation independence, a hallmark of enhancers

• Linking phosphorylation to enhancer function:

  • Analyze FOXO3 phosphorylation status at enhancers versus promoters

  • Track phospho-FOXO3a (Ser253) ChIP signal at enhancer regions

  • Correlate FOXO3 phosphorylation with RNA polymerase II recruitment

• Chromatin conformation techniques:

  • 4C-sequencing to identify chromatin loops between FOXO3 bound enhancers and target genes

  • "Pre-existing enhancers and promoter–enhancer loops are responsible for differential responses to FOXO activity"

What experimental approaches can elucidate the relationship between FOXO3a phosphorylation and its protein stability?

To investigate phosphorylation-dependent FOXO3a stability:

• Cycloheximide chase experiments:

  • Treat cells with cycloheximide (50 μg/ml) to inhibit protein synthesis

  • Monitor phospho-FOXO3a (Ser253) degradation over 0-24 hours

  • Focus on early time points (0, 5, 10, 15, and 30 minutes) as "phosphorylated FOXO3 found in the cytoplasm can be rapidly degraded"

  • Include controls like STAT3 to confirm protein degradation occurs

• Ubiquitination assays:

  • Immunoprecipitate FOXO3a and blot for ubiquitin

  • Compare ubiquitination of wild-type versus phospho-mutant FOXO3a (S253A)

  • "PP2A is required for the reactivation of FoxO3a by promoting its translocation to the nucleus"

• Proteasome inhibition studies:

  • Treat cells with MG132 (proteasome inhibitor)

  • Compare phospho-FOXO3a (Ser253) levels before and after treatment

  • "Adenovirus E1A stabilizes FoxO3a by inducing the expression of PP2A/C, which suppresses βTrCP-mediated degradation of FoxO3a"

• Phospho-mutant analysis:

  • Generate S253A (non-phosphorylatable) and S253D/E (phospho-mimetic) mutants

  • Compare protein half-lives of these mutants to wild-type FOXO3a

  • Analyze subcellular localization and transcriptional activity

What methodological considerations are important when studying the effects of FOXO3a phosphorylation on its DNA binding capabilities?

To properly assess how phosphorylation affects FOXO3a-DNA interactions:

• DNA binding assays:

  • Fluorescence anisotropy assays using "a DNA oligonucleotide incorporating the FOXO3 consensus sequence (5′-CGCATCCTATGTAAACAACTCGAGTC-3′)"

  • EMSA (electrophoretic mobility shift assay) with labeled FOXO3a binding sites

  • Compare binding of dephosphorylated versus phosphorylated FOXO3a

• Binding site selection:

  • FOXO3a recognizes two consensus sequences:

    1. 5'-GTAAA(T/C)AA-3' (Daf-16 family member-binding element)

    2. 5'-(C/A)(A/C)AAA(C/T)AA-3' (insulin-responsive sequence, IRE)

  • Design experiments to test if phosphorylation affects binding preference

• Structural considerations:

  • Crystal structure reveals "the recognition helix H3 docked perpendicular to the major groove making extensive contacts with the DNA"

  • Phosphorylation at S253 might influence this interaction

  • Use FOXO3a mutant constructs to map interaction domains:

    • FL (full length, 1–673)

    • M1 (mutant with deletion in N terminus, 151–673)

    • M2 (mutant with deletion in N terminus and Forkhead domain, 244–673)

• Competitive binding assays:

  • Test if NF-κB RelA binding to FOXO3a interferes with DNA binding

  • "FOXO3 participates in regulation of cellular functions through additional mechanisms"

  • Consider the influence of other FOXO3a binding partners on DNA interaction