Phospho-FOXO1 (Ser319) Antibody

What is FOXO1 and what is the significance of its phosphorylation at Serine 319?

FOXO1 (Forkhead box protein O1) is a key transcription factor belonging to the forkhead family that regulates various cellular processes including cell cycle regulation, apoptosis, and metabolism. It serves as a central regulator of metabolism in several cell types.

Phosphorylation of FOXO1 at Serine 319 is particularly significant as it modulates the protein's transcriptional activity and cellular localization. This phosphorylation event is part of the regulatory mechanism that controls FOXO1's ability to influence gene expression related to insulin sensitivity, oxidative stress, and longevity . When phosphorylated at Ser319, FOXO1 typically undergoes nuclear export, which inhibits its transcriptional activity .

What cellular processes and pathways involve Phospho-FOXO1 (Ser319)?

Phospho-FOXO1 (Ser319) is involved in several critical cellular pathways:

• Insulin signaling pathway: Insulin-induced phosphorylation affects FOXO1 localization and activity

• Cell cycle regulation and apoptotic pathways

• Glucose metabolism and insulin sensitivity

• Stress response pathways, including oxidative stress

• T cell differentiation and immune function regulation

• Cancer-related signaling pathways

Recent research has identified that the GLK-IKKβ signaling pathway triggers phosphorylation of FOXO1 at Ser319, leading to its nuclear export . This phosphorylation has been implicated in Treg differentiation inhibition, which has important implications for immune system regulation .

How does phosphorylation at Ser319 affect FOXO1 function compared to other phosphorylation sites?

Phosphorylation at Ser319 specifically affects FOXO1 function in the following ways:

Specifically, phosphorylation at Ser319 by IKKβ leads to FOXO1 nuclear export and decreased transcriptional activity . This is distinct from oxidative stress-induced phosphorylation at Ser212, which causes nuclear translocation and retention .

What are the recommended applications for Phospho-FOXO1 (Ser319) antibody and their respective dilutions?

Based on manufacturer specifications, Phospho-FOXO1 (Ser319) antibody can be used in various applications with the following recommended dilutions:

It's essential to note that optimal dilutions may vary depending on the specific antibody product and experimental conditions. Titration is recommended to determine the optimal concentration for each application and sample type .

How should researchers validate the specificity of the Phospho-FOXO1 (Ser319) antibody?

To validate the specificity of Phospho-FOXO1 (Ser319) antibody, researchers should:

• Use appropriate positive controls: NIH-3T3 cells for Western blot, human breast carcinoma for IHC, and HeLa cells for ICC/IF as suggested by manufacturers .

• Employ phosphorylation state-specific validation: Compare antibody reactivity in:

  • Untreated cells (low phosphorylation)

  • Insulin-stimulated cells (increased phosphorylation)

  • Cells treated with phosphatase inhibitors

• Utilize peptide competition assays: Pre-incubate the antibody with the phosphorylated peptide used as immunogen (typically peptide sequence around phosphorylation site of serine 319 (T-S-S(p)-N-A) ).

• Perform phospho-deficient mutant analysis: Test the antibody against cells expressing FoxO1 (S319A) mutant, which should show no reactivity if the antibody is specific .

• Cross-reactivity assessment: Verify that the antibody does not cross-react with unphosphorylated FOXO1 or with other phosphorylated FOXO family members (e.g., FOXO3a) .

What are the optimal sample preparation methods for detecting Phospho-FOXO1 (Ser319)?

For optimal detection of Phospho-FOXO1 (Ser319), sample preparation should be tailored to the specific application:

For Western Blot:

• Rapidly harvest cells to preserve phosphorylation state

• Lyse cells in buffer containing phosphatase inhibitors (critical)

• Use freshly prepared samples when possible

• For protein extraction from tissues, flash-freeze samples immediately after collection

For Immunocytochemistry/Immunofluorescence:

• PFA fixation (4%) followed by methanol permeabilization has been demonstrated to produce strong signals and the expected localization pattern

• Avoid Triton X-100 permeabilization as it may not preserve the phospho-epitope effectively

For Immunohistochemistry:

• Formalin-fixed paraffin-embedded tissues should be subjected to antigen retrieval

• Use heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

• Include phosphatase inhibitors in all buffers when possible

How can researchers study the dynamic regulation of FOXO1 Ser319 phosphorylation in response to different stimuli?

To study dynamic regulation of FOXO1 Ser319 phosphorylation:

• Time-course experiments: Monitor phosphorylation levels at different time points following stimulus application (e.g., insulin, oxidative stress, growth factors).

• Kinase inhibition studies: Use specific inhibitors for:

  • IKKβ (identified as directly phosphorylating FOXO1 at Ser319)

  • AKT pathway inhibitors to distinguish between direct and indirect phosphorylation events

• Co-immunoprecipitation assays: To detect the physical interaction between FOXO1 and kinases (like IKKβ) or phosphatases under different conditions.

• Live-cell imaging:

  • Use fluorescently tagged FOXO1 constructs combined with phospho-specific antibodies

  • Monitor subcellular localization changes in real-time following stimulation

• Phospho-proteomic approaches: Mass spectrometry analysis to identify multiple phosphorylation events simultaneously. The MS/MS fragmentation spectra of trypsin-digested FOXO1 peptides can reveal the phosphorylation status at Ser319 .

• In vitro kinase assays: Using purified GST-tagged FOXO1 and Flag-tagged IKKβ to confirm direct phosphorylation .

What are the technical challenges in detecting Phospho-FOXO1 (Ser319) in different experimental systems?

Researchers face several technical challenges when detecting Phospho-FOXO1 (Ser319):

• Phosphorylation instability: Phosphorylation can be rapidly lost during sample handling due to phosphatase activity. Always use fresh phosphatase inhibitors.

• Signal-to-noise ratio: Background issues can occur, particularly in immunostaining applications. Proper blocking and antibody titration are critical.

• Tissue-specific expression levels: FOXO1 expression varies across tissues, requiring optimization for each tissue type.

• Cross-reactivity concerns: Some antibodies may detect other phosphorylated FoxO family members. Validation with phospho-deficient mutants is recommended.

• Fixation-dependent epitope masking: For microscopy applications, different fixation methods can mask the phospho-epitope. PFA fixation followed by methanol permeabilization has been shown to work effectively .

• Nuclear-cytoplasmic shuttling: Since phosphorylation at Ser319 affects subcellular localization, careful subcellular fractionation is required when analyzing total cell lysates.

• Stimulus timing: The transient nature of phosphorylation events requires precise timing of stimulation and sample collection.

How does the Phospho-FOXO1 (Ser319) status correlate with pathological conditions?

Phosphorylation of FOXO1 at Ser319 has been implicated in several pathological conditions:

• Diabetes and Metabolic Disorders:

  • Altered phosphorylation contributes to insulin resistance

  • FOXO1 acts synergistically with ATF4 to suppress osteocalcin/BGLAP activity, increasing glucose levels and triggering glucose intolerance and insulin insensitivity

• Cancer:

  • Dysregulated FOXO1 phosphorylation is observed in multiple cancer types

  • Particularly relevant in human breast carcinoma, where it's used as a positive control for immunohistochemistry

  • The nuclear-cytoplasmic shuttling controlled by Ser319 phosphorylation affects tumor suppressor functions

• Autoimmune Disorders:

  • Recent research demonstrates that IKKβ-mediated phosphorylation of FOXO1 at Ser319 inhibits Treg differentiation

  • This mechanism may contribute to immune dysregulation in autoimmune conditions

• Aging-related Disorders:

  • FOXO1 activity influences longevity pathways

  • Altered phosphorylation patterns are observed in aging tissues

What is the relationship between FOXO1 Ser319 phosphorylation and its interaction with other proteins?

Phosphorylation at Ser319 affects FOXO1's interactome in several significant ways:

• 14-3-3 Protein Binding:

  • While Thr24 phosphorylation is the primary site for 14-3-3 binding, Ser319 phosphorylation by IKKβ contributes to creating the full binding interface

  • This interaction is critical for nuclear export and cytoplasmic retention

• Transcriptional Complex Formation:

  • Phosphorylation disrupts interactions with DNA and co-activators

  • In osteoblasts, nuclear FOXO1 colocalizes with ATF4 and RUNX2; phosphorylation at Ser319 would disrupt these interactions

• Ubiquitin-Proteasome Pathway:

  • Cytoplasmic FOXO1 following Ser319 phosphorylation becomes susceptible to ubiquitination and degradation

• IKKβ Interaction:

  • IKKβ directly phosphorylates FOXO1 at Ser319, with enhanced activity when IKKβ itself is phosphorylated at Ser733

  • This has been confirmed through in vitro kinase assays using purified proteins

• Competition with Other Post-translational Modifications:

  • Ser319 phosphorylation may compete with other modifications like methylation that promote nuclear retention

How can researchers differentiate between direct and indirect effects on FOXO1 Ser319 phosphorylation in complex signaling networks?

To differentiate between direct and indirect phosphorylation mechanisms:

• In vitro kinase assays: Use purified components to demonstrate direct phosphorylation, as shown with GST-tagged FOXO1 and Flag-tagged IKKβ or IKKβ kinase-dead (K44M) mutant proteins .

• Phospho-mutant studies: Compare wild-type FOXO1 with phospho-deficient (S319A) and phospho-mimetic (S319E) mutants to isolate the effects of this specific phosphorylation site .

• Proximity ligation assays (PLA): Detect direct protein-protein interactions between FOXO1 and candidate kinases in intact cells. The search results mention PLA being used to demonstrate interactions in the GLK-PKCθ-IKKβ pathway .

• Pathway inhibition hierarchy:

  • Systematically inhibit components of signaling cascades

  • Determine if Ser319 phosphorylation is affected by upstream or parallel pathway inhibition

  • Compare with other FOXO1 phosphorylation sites to identify pathway-specific patterns

• Kinase substrate trap mutants: Use catalytically inactive "substrate-trapping" mutants of candidate kinases to capture and identify physiological substrates.

• Genetic models: Use knockout or knockdown models of specific pathway components to establish hierarchy and necessity in the signaling cascade leading to FOXO1 Ser319 phosphorylation.

What are common problems in Phospho-FOXO1 (Ser319) antibody-based experiments and how can they be overcome?

How should researchers validate experimental results obtained with Phospho-FOXO1 (Ser319) antibodies?

For robust validation of results:

• Use multiple detection methods:

  • Combine Western blot with immunofluorescence or immunohistochemistry

  • Verify cellular localization changes expected with phosphorylation status

• Functional validation:

  • Correlate phosphorylation with expected downstream effects (e.g., target gene expression)

  • Use phospho-deficient (S319A) and phospho-mimetic (S319E) FOXO1 mutants

• Pharmacological validation:

  • Use known modulators of FOXO1 phosphorylation (e.g., insulin stimulation increases phosphorylation)

  • Include appropriate kinase inhibitors (e.g., IKKβ inhibitors) to confirm pathway specificity

• Genetic knockdown/knockout controls:

  • Include FOXO1 knockdown/knockout samples as negative controls

  • Use cells from genetic models with altered kinase expression (e.g., IKKβ knockout)

• Cross-validation with mass spectrometry:

  • Confirm phosphorylation status using phospho-proteomics approaches

  • MS/MS fragmentation spectra can definitively identify phosphorylated residues

• Reproducibility across cell types and conditions:

  • Test in multiple relevant cell types

  • Verify consistent response patterns to stimuli