Phospho-FOXO4 (Ser197) Antibody

What is FOXO4 and what is the significance of its phosphorylation at Ser197?

FOXO4 (Forkhead box O4) is a transcription factor belonging to the O class of forkhead/winged helix family of transcription factors. It is encoded by the FOXO4 gene located on the X chromosome and is also known as AFX1 or MLLT7. FOXO4 is predominantly expressed in skeletal muscle and adipose tissue .

Phosphorylation at Ser197 is particularly significant because:

• It occurs within the DNA binding domain (DBD) of FOXO4

• It regulates FOXO4's transcriptional activity

• It mediates binding to 14-3-3 proteins, which affects nuclear-cytoplasmic shuttling

• It plays a crucial role in cellular responses to insulin/IGF1 signaling

This specific phosphorylation site serves as a critical regulatory mechanism for FOXO4's role in controlling cell cycle progression, apoptosis, and stress resistance, making it an important target for research in cancer, diabetes, and aging.

What experimental applications can Phospho-FOXO4 (Ser197) antibody be used for?

Phospho-FOXO4 (Ser197) antibodies have been validated for multiple research applications:

When performing these applications, researchers should optimize antibody dilutions according to manufacturer recommendations and include appropriate positive and negative controls to ensure specificity for phosphorylated FOXO4 at Ser197 .

How specific is the Phospho-FOXO4 (Ser197) antibody, and how can this be verified?

The specificity of Phospho-FOXO4 (Ser197) antibodies is typically very high, with several verification methods confirming this:

• These antibodies selectively detect FOXO4 only when phosphorylated at Serine 197, not recognizing non-phosphorylated forms

• Specificity is achieved through affinity purification via sequential chromatography on phospho- and non-phospho-peptide affinity columns

• The antibodies are generated using synthetic phosphopeptides derived from human FOXO4 around the phosphorylation site of Ser197

Researchers can verify specificity by:

• Using phosphatase treatments as negative controls

• Employing blocking peptides (phosphorylated vs. non-phosphorylated) in competitive binding experiments

• Testing against samples where FOXO4 phosphorylation is induced or inhibited

• Using FOXO4 knockout or knockdown samples as negative controls

The antibody has demonstrated high specificity in ELISA assays when comparing reactivity against phospho vs. non-phospho peptides .

What signaling pathways regulate FOXO4 phosphorylation at Ser197?

Multiple signaling pathways converge on FOXO4 Ser197 phosphorylation:

Akt/PKB Pathway:

• Activation of insulin/IGF1 receptors leads to PI3K-mediated Akt activation

• Activated Akt can phosphorylate FOXO4 at Ser197

• This phosphorylation requires functional LKB1, as depletion of LKB1 in H1650 cells resulted in decreased phosphorylation of FOXO4 at Ser197

RSK2 (Ribosomal S6 kinase 2) Pathway:

• RSK2 directly phosphorylates FOXO4 at Ser197 and Ser262

• RSK2 physically interacts with FOXO4 via its DNA binding domain (DBD) spanning amino acids 100-180

• This interaction was confirmed through pull-down assays and in vitro kinase assays

LKB1-dependent mechanisms:

• LKB1 is necessary for Akt-mediated phosphorylation of FOXO4 at Ser197

• This provides a link between tumor suppressor pathways and FOXO4 regulation

Understanding these interconnected pathways is crucial for interpreting experiments targeting FOXO4 phosphorylation status.

What are the best protocols for detecting Phospho-FOXO4 (Ser197) in Western blots?

For optimal Western blot detection of Phospho-FOXO4 (Ser197):

Sample preparation:

• Use phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers

• Maintain samples at cold temperatures during processing

• Use fresh samples when possible to prevent phosphorylation loss

Western blot procedure:

• Separate proteins on 10% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% BSA in TBST (not milk, which contains phosphatases)

• Incubate with Phospho-FOXO4 (Ser197) antibody at manufacturer's recommended dilution (typically 1:1000)

• Expected bands: 55 and 70 kDa

• For detection, use HRP-conjugated secondary antibodies and ECL detection systems

Controls to include:

• Positive control: Insulin-stimulated cells (activates Akt pathway)

• Negative control: Phosphatase-treated lysates

• Loading control: GAPDH or β-actin

Troubleshooting tips:

• If background is high, increase blocking time or use a different blocking reagent

• If signal is weak, increase antibody concentration or extend incubation time

• For reproducible results, standardize protein loading and transfer conditions

How can I validate the specificity of Phospho-FOXO4 (Ser197) antibody in my experimental system?

To thoroughly validate Phospho-FOXO4 (Ser197) antibody specificity in your system:

Experimental validation approaches:

• Pharmacological treatments:

  • Compare samples treated with PI3K/Akt inhibitors (e.g., LY294002, Wortmannin) versus activators (e.g., insulin, IGF-1)

  • RSK2 inhibitors (e.g., BI-D1870) can also be used to reduce Ser197 phosphorylation

• Genetic approaches:

  • Use FOXO4 knockdown or knockout cells as negative controls

  • Express wild-type versus S197A mutant FOXO4 (cannot be phosphorylated at this site)

  • Compare LKB1-proficient versus LKB1-deficient cells

• Biochemical validation:

  • Treat lysates with lambda phosphatase to remove phosphorylation

  • Perform peptide competition assays using phospho-Ser197 peptides

  • Compare reactivity with other phospho-FOXO4 antibodies targeting different sites

• Multiple detection methods:

  • Confirm results using both immunoblotting and immunohistochemistry/immunofluorescence

  • Validate with ELISA-based quantification methods

These validation steps ensure that any observed signals genuinely represent phosphorylated FOXO4 at Ser197 rather than non-specific binding or cross-reactivity.

How does RSK2-mediated phosphorylation of FOXO4 at Ser197 differ from Akt-mediated phosphorylation?

The differences between RSK2 and Akt-mediated phosphorylation of FOXO4 at Ser197 present important mechanistic distinctions:

Structural interactions:

• RSK2 specifically interacts with FOXO4's DNA binding domain (DBD) spanning amino acids 100-180

• RSK2 recognizes both RxRxxS/T and RxxS/T phosphorylation motifs in FOXO4

• RSK2 simultaneously phosphorylates FOXO4 at both Ser197 and Ser262

• Akt primarily recognizes RxRxxS/T motifs

Upstream regulation:

• RSK2 is primarily activated through the MAPK/ERK pathway

• Akt is activated through the PI3K pathway downstream of insulin/IGF1 signaling

• LKB1 is specifically required for Akt-mediated, but not necessarily RSK2-mediated, phosphorylation

Functional consequences:

This dual regulation provides cells with multiple ways to modulate FOXO4 activity in response to different stimuli, which may explain the complex roles of FOXO4 in various cellular processes.

What is the role of LKB1 in regulating FOXO4 phosphorylation at Ser197?

LKB1 (Liver Kinase B1) plays a crucial but incompletely understood role in regulating FOXO4 phosphorylation:

Experimental evidence:

• Depletion of LKB1 in H1650 cells results in decreased phosphorylation of multiple Akt substrates, including FOXO4 at Ser197

• This effect appears to be part of a broader impact on Akt signaling, as other Akt substrates including Bad (Ser136), FoxO1 (Ser319), and Gsk3β (Ser9) show similarly reduced phosphorylation

Mechanistic models:

• LKB1 may directly or indirectly participate in the phosphorylation of Akt target proteins

• Direct interaction between LKB1 and Akt has not been previously demonstrated

• LKB1 may exert its effects through downstream substrates, potentially including AMPK

• LKB1 has at least 12 known substrates whose functions remain poorly characterized

Research implications:

• The requirement for LKB1 in Akt-mediated phosphorylation events represents an important intersection between tumor suppressor signaling and metabolic regulation

• This connection may explain some of the pleiotropic effects of LKB1 loss in cancer and metabolic disorders

• Further research is needed to determine whether LKB1 or its downstream targets directly participate in the phosphorylation of FOXO4

Understanding this pathway has implications for therapeutic approaches targeting FOXO4 regulation in diseases where LKB1 function is compromised.

How can I design experiments to study the dynamic regulation of FOXO4 Ser197 phosphorylation?

To effectively study dynamic FOXO4 Ser197 phosphorylation:

Time-course experiments:

• Stimulate cells with insulin/IGF1 or growth factors and collect samples at multiple timepoints (0, 5, 15, 30, 60, 120 min)

• Monitor both phosphorylation status and subcellular localization simultaneously

• Use cell-based ELISA kits for high-throughput quantitative assessment

Signal pathway modulation:

• Compare effects of multiple pathway inhibitors:

  • PI3K/Akt inhibitors (LY294002, Wortmannin)

  • MEK/ERK inhibitors (U0126, PD98059) affecting RSK2

  • mTOR inhibitors (rapamycin, Torin1)

  • LKB1/AMPK modulators (metformin, compound C)

Advanced imaging approaches:

• Utilize FRET-based biosensors incorporating FOXO4 to visualize phosphorylation events in real-time

• Perform fluorescence recovery after photobleaching (FRAP) to assess how phosphorylation affects FOXO4 mobility

Molecular techniques:

• Generate phosphomimetic (S197D) and phosphodeficient (S197A) FOXO4 mutants to study functional consequences

• Employ CRISPR/Cas9 gene editing to create endogenous FOXO4 mutations

• Use proximity ligation assays to detect interactions between phosphorylated FOXO4 and binding partners like 14-3-3 proteins

Data analysis approaches:

• Apply systems biology modeling to integrate phosphorylation data with transcriptional outcomes

• Use multiplexed detection methods to simultaneously assess multiple phosphorylation sites on FOXO4

• Correlate phosphorylation levels with functional readouts like target gene expression

These approaches collectively provide a comprehensive view of FOXO4 Ser197 phosphorylation dynamics.

What are the technical considerations for troubleshooting specificity issues with Phospho-FOXO4 (Ser197) antibodies?

When encountering specificity issues with Phospho-FOXO4 (Ser197) antibodies:

Common problems and solutions:

• Cross-reactivity with other phospho-proteins:

  • Verify that bands appear at the expected molecular weight (55-70 kDa)

  • Use FOXO4 knockout or knockdown controls

  • Perform peptide competition assays with phospho-Ser197 peptides versus other phosphopeptides

  • Consider using antibodies from multiple vendors or with different epitopes to confirm results

• High background in immunostaining:

  • Optimize blocking conditions (try 5% BSA, serum-free protein blocks, or commercial blockers)

  • Test longer/more stringent washing steps

  • Reduce primary antibody concentration

  • Use a more specific secondary antibody

  • Include phosphopeptide blocking controls

• Inconsistent results between experiments:

  • Standardize lysate preparation with consistent phosphatase inhibitors

  • Prepare fresh phosphatase inhibitor cocktails for each experiment

  • Process samples quickly at cold temperatures

  • Avoid repeated freeze-thaw cycles

  • Compare results from multiple techniques (WB, ELISA, IHC)

• False negatives (no signal detection):

  • Confirm phosphorylation status by using appropriate positive controls (insulin-stimulated cells)

  • Verify that the antibody recognizes your species' FOXO4 (check sequence homology)

  • Test multiple antibody dilutions and incubation conditions

  • Consider enriching phosphoproteins before analysis

• Validation using complementary approaches:

  • Phosphorylation-specific mass spectrometry analysis

  • In vitro kinase assays with purified components

  • Parallel analysis with pan-FOXO4 antibodies to confirm protein presence

These troubleshooting strategies help ensure reliable and reproducible results when working with phospho-specific antibodies.