Phospho-FER (Tyr402) Antibody

What is FER kinase and why is phosphorylation at Tyr402 significant?

FER is a non-receptor tyrosine kinase belonging to the FPS/FES family that regulates cell-cell adhesion and mediates signaling from the cell surface to the cytoskeleton via growth factor receptors . It is ubiquitously expressed and found in both cytoplasmic and nuclear compartments, where it associates with the chromatin fraction . FER functions as a downstream effector of multiple cell surface receptors including EGFR, KIT, PDGFRA, and PDGFRB, playing crucial roles in the regulation of actin cytoskeleton, microtubule assembly, lamellipodia formation, cell migration, and chemotaxis .

Phosphorylation at Tyrosine 402 (Tyr402) represents a critical activation marker of FER kinase. This site is autophosphorylated during kinase activation and serves as an important regulatory mechanism controlling FER's catalytic activity and substrate specificity . Detection of phosphorylation at this specific residue provides researchers with a precise method to monitor FER activation status in various experimental contexts.

What are the recommended applications for Phospho-FER (Tyr402) Antibody?

Phospho-FER (Tyr402) antibodies are validated for multiple research applications with specific recommended dilution ranges:

The antibody specifically detects endogenous levels of FER protein only when phosphorylated at Tyr402, making it valuable for studying activation states of this kinase in various experimental conditions . When implementing these techniques, researchers should optimize dilutions based on their specific experimental system and sample types.

How should researchers validate antibody specificity in their experimental systems?

Validation of Phospho-FER (Tyr402) antibody specificity is critical for ensuring experimental rigor. Recommended validation strategies include:

• Kinase-dead mutant controls: Compare phospho-tyrosine patterns in cells transfected with wild-type FER versus kinase-dead mutant (K591R) FER . The absence of signal in the kinase-dead mutant samples confirms specificity.

• Phosphatase treatment: Treat half of your sample with lambda phosphatase prior to immunoblotting. Loss of signal after phosphatase treatment confirms phospho-specificity.

• Peptide competition: Pre-incubate the antibody with the immunizing phosphopeptide (derived from the human FER around Tyr402 at amino acid range 371-420) . Signal reduction indicates specificity for the target epitope.

• siRNA/shRNA knockdown: Deplete endogenous FER and observe corresponding reduction in phospho-signal.

These validation steps are particularly important when investigating new cell types or experimental conditions where antibody performance has not been previously established.

How can researchers use Phospho-FER (Tyr402) Antibody to identify novel FER substrates?

Identifying novel FER substrates requires a strategic approach combining immunoprecipitation, mass spectrometry, and validation techniques. Based on established research methodologies:

• Enrichment and identification strategy:

  • Transfect cells with wild-type FER and kinase-dead FER (K591R)

  • Enrich tyrosine-phosphorylated proteins using anti-phosphotyrosine antibody (4G10)

  • Perform mass spectrometry analysis on the enriched proteins

  • Compare phosphoprotein profiles between wild-type and kinase-dead FER to identify potential substrates

• Validation methodology:

  • Confirm direct phosphorylation using in vitro kinase assays with purified FER kinase domain (e.g., GST-tagged human FER tyrosine kinase domain 541-822aa)

  • Perform site-directed mutagenesis to identify specific tyrosine residues phosphorylated by FER

  • Validate physiological relevance through co-immunoprecipitation and functional assays

This approach has successfully identified novel substrates such as IRS4, which was found to be phosphorylated by FER at multiple tyrosine residues (Y656, Y779, Y828, Y921) leading to PI3K-AKT pathway activation .

What is known about the role of FER phosphorylation in cancer signaling pathways?

FER phosphorylation plays significant roles in cancer signaling networks, particularly in ovarian cancer:

• PI3K-AKT pathway activation:

  • FER directly phosphorylates IRS4 at multiple tyrosine residues (Y656, Y779, Y828, Y921)

  • Phosphorylated IRS4 recruits PIK3R2, activating the PI3K-AKT signaling pathway

  • This activation promotes tumorigenesis in ovarian cancer cells

• Receptor tyrosine kinase signaling:

  • FER regulates MET receptor tyrosine kinase signaling through phosphorylation of Y1349 and Y1356 residues

  • FER maintains plasma membrane distribution of MET, delaying protein-tyrosine phosphatase activity

  • This spatial regulation enhances growth factor signaling duration and intensity

• NF-κB pathway involvement:

  • FER acts downstream of EGFR to promote NF-κB activation and cell proliferation

  • This mechanism contributes to cancer cell survival and tumor progression

These findings suggest that monitoring FER phosphorylation status using Phospho-FER (Tyr402) antibody provides valuable insights into cancer signaling mechanisms and potential therapeutic targets.

How does FER kinase interact with other signaling proteins in experimental systems?

FER kinase interacts with multiple signaling proteins, forming complex regulatory networks:

• Growth factor receptor interactions:

  • FER functions downstream of receptor tyrosine kinases including EGFR, KIT, PDGFRA, and PDGFRB

  • It mediates signal transduction from these receptors to regulate cellular processes

• Adapter protein interactions:

  • FER phosphorylates IRS4, creating binding sites for signaling proteins like PIK3R2

  • This interaction facilitates recruitment of PI3K complex components

• Cytoskeletal regulators:

  • FER modulates actin cytoskeleton dynamics and microtubule assembly

  • It regulates lamellipodia formation, cell adhesion, and migration through these interactions

• Phosphatase regulation:

  • FER influences the activity of protein-tyrosine phosphatases through spatial regulation

  • This mechanism extends the duration of growth factor signaling

Researchers can use co-immunoprecipitation studies with Phospho-FER (Tyr402) antibody to investigate these interaction networks and their dynamics under various experimental conditions.

What are the optimal protocols for immunoprecipitation and western blotting using Phospho-FER (Tyr402) Antibody?

For optimal results when using Phospho-FER (Tyr402) Antibody in immunoprecipitation and western blotting:

Immunoprecipitation Protocol:

• Lyse cells in modified RIPA buffer (50mM Tris-HCl pH7.4, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 50mM NaF, 1mM Na₃VO₄, 10% glycerol, protease inhibitor cocktail)

• Incubate lysates at 4°C for 30 minutes

• Clear lysates by centrifugation at 12,000g for 15 minutes

• Determine protein concentration using Bradford assay

• For each sample, use 500-1000 μg of total protein

• Add 2-5 μg of Phospho-FER (Tyr402) antibody

• Incubate overnight at 4°C with gentle rotation

• Add protein A/G beads and incubate for 2-4 hours at 4°C

• Wash beads 4-5 times with lysis buffer

• Elute proteins with SDS sample buffer and analyze by western blotting

Western Blotting Protocol:

• Resolve proteins on 8-10% SDS-PAGE gel

• Transfer to PVDF membrane at 100V for 90 minutes

• Block membrane with 5% BSA in TBST for 1 hour at room temperature

• Incubate with Phospho-FER (Tyr402) antibody at 1:1000 dilution in 5% BSA/TBST overnight at 4°C

• Wash 3x with TBST for 10 minutes each

• Incubate with HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature

• Wash 3x with TBST for 10 minutes each

• Develop using ECL reagent and capture images

Including appropriate controls is essential for result interpretation. The antibody formulation (liquid in PBS containing 50% Glycerol, 0.5% BSA and 0.02% Sodium Azide) should be stored at -20°C to maintain activity .

How can researchers induce and detect FER phosphorylation in experimental systems?

Several approaches can be used to induce and detect FER phosphorylation in experimental systems:

Induction Methods:

• Growth factor stimulation:

  • Treat serum-starved cells with growth factors that activate receptor tyrosine kinases upstream of FER (e.g., EGF, PDGF, HGF)

  • Typical concentrations: EGF (50-100 ng/ml), PDGF (25-50 ng/ml), HGF (20-40 ng/ml)

  • Optimal time points: 5, 15, 30, 60 minutes

• Overexpression systems:

  • Transiently transfect cells with wild-type FER expression constructs

  • This leads to autophosphorylation of FER at Tyr402 due to increased local concentration

• Phosphatase inhibition:

  • Treat cells with tyrosine phosphatase inhibitors (e.g., sodium orthovanadate at 1-2 mM)

  • This prevents dephosphorylation of FER, increasing detectable phospho-FER levels

Detection Methods:

• Western blotting:

  • Use Phospho-FER (Tyr402) antibody at 1:500-1:2000 dilution

  • Include total FER antibody to normalize phosphorylation levels

• Immunofluorescence:

  • Use Phospho-FER (Tyr402) antibody at 1:50-1:200 dilution

  • Co-stain with markers for relevant cellular compartments

• Multiplex analysis:

  • Combine Phospho-FER (Tyr402) detection with other phospho-specific antibodies to analyze pathway activation

These approaches enable researchers to study the dynamics of FER activation in response to various stimuli and in different cellular contexts.

What controls should be included when analyzing FER phosphorylation?

When analyzing FER phosphorylation, researchers should include several controls to ensure result validity:

Essential Controls for Phospho-FER (Tyr402) Analysis:

• Positive controls:

  • Cells overexpressing wild-type FER (leads to autophosphorylation)

  • Cells treated with growth factors known to activate FER (e.g., EGF, PDGF)

• Negative controls:

  • Kinase-dead FER mutant (K591R) expressing cells

  • FER-depleted cells (siRNA or CRISPR knockout)

  • Phosphatase-treated samples

• Antibody specificity controls:

  • Peptide competition with the immunizing phosphopeptide

  • Secondary antibody-only control

• Pathway context controls:

  • Inhibitors of upstream kinases to confirm pathway-specific activation

  • Phosphorylation analysis of known FER substrates (e.g., IRS4)

• Loading and normalization controls:

  • Total FER protein detection

  • Housekeeping proteins (β-actin, β-tubulin)

Including these controls helps distinguish specific FER phosphorylation from background signals and validates the physiological relevance of observed phosphorylation events in experimental systems.

How is Phospho-FER (Tyr402) Antibody used to study cancer signaling pathways?

Phospho-FER (Tyr402) Antibody serves as a valuable tool for investigating cancer signaling pathways, particularly in ovarian cancer research:

• Oncogenic signaling pathway analysis:

  • Researchers use this antibody to monitor FER activation status in cancer cell lines and patient samples

  • Studies have demonstrated that FER activation promotes AKT signaling and tumorigenesis in ovarian cancer cells

  • The antibody helps track FER-mediated phosphorylation of downstream targets like IRS4

• Substrate identification and validation:

  • The antibody has been instrumental in confirming FER as the kinase responsible for specific tyrosine phosphorylation events

  • Mass spectrometry combined with immunoprecipitation using phospho-tyrosine antibodies identified 2298 candidate FER substrates

  • Subsequent validation with Phospho-FER (Tyr402) Antibody confirmed direct phosphorylation relationships

• Therapeutic target assessment:

  • Researchers use the antibody to evaluate FER as a potential therapeutic target

  • Monitoring changes in Tyr402 phosphorylation helps assess the efficacy of FER kinase inhibitors

  • This approach aids in developing strategies to target FER-dependent signaling in cancer

These applications demonstrate the utility of Phospho-FER (Tyr402) Antibody in unraveling complex cancer signaling networks and identifying potential therapeutic interventions.

What insights has Phospho-FER (Tyr402) Antibody provided about immune system regulation?

The Phospho-FER (Tyr402) Antibody has contributed to understanding FER's role in immune regulation:

• Leukocyte recruitment regulation:

  • FER has been linked to the regulation of leukocyte recruitment during the innate immune response

  • Phospho-FER (Tyr402) Antibody helps track FER activation status in immune cells under various stimulation conditions

  • This provides insights into how FER phosphorylation correlates with immune cell mobilization and function

• Cytokine signaling:

  • FER responds to cytokine stimulation in immune cells

  • The antibody enables researchers to monitor FER activation in response to specific cytokines

  • This helps elucidate the role of FER in cytokine-mediated immune signaling networks

• Cell adhesion in immune context:

  • FER regulates cell-cell adhesion, which is crucial for immune cell interactions

  • Phospho-FER (Tyr402) Antibody allows tracking of FER activation during immune cell adhesion events

  • This contributes to understanding how FER modulates immune cell interactions and migration

These applications highlight how Phospho-FER (Tyr402) Antibody serves as a valuable tool for investigating FER's diverse roles in immune regulation and response.

How can Phospho-FER (Tyr402) Antibody be used to study receptor tyrosine kinase signaling?

Phospho-FER (Tyr402) Antibody provides valuable insights into receptor tyrosine kinase (RTK) signaling mechanisms:

• Downstream effector analysis:

  • FER functions downstream of multiple RTKs including EGFR, KIT, PDGFRA, and PDGFRB

  • The antibody allows researchers to track FER activation following RTK stimulation

  • This helps map signaling cascades and temporal dynamics of RTK-induced FER activation

• Spatial regulation studies:

  • FER maintains plasma membrane distribution of MET receptor tyrosine kinase

  • Researchers use Phospho-FER (Tyr402) Antibody in conjunction with subcellular fractionation or immunofluorescence to study this spatial regulation

  • This approach has revealed how FER delays protein-tyrosine phosphatase activity on RTKs

• Cross-talk investigation:

  • FER mediates signaling between different RTK pathways

  • The antibody helps identify conditions where FER serves as an integration point for multiple RTK signals

  • This contributes to understanding complex signaling networks in normal and disease states

• Temporal dynamics analysis:

  • Using Phospho-FER (Tyr402) Antibody at different time points after RTK stimulation reveals the kinetics of FER activation

  • This temporal information helps understand how FER contributes to both immediate and sustained RTK signaling responses

These applications demonstrate how Phospho-FER (Tyr402) Antibody serves as a critical tool for dissecting complex RTK signaling networks and their dysregulation in disease.