Phospho-ITCH (Y420) Antibody

What is ITCH and what role does Y420 phosphorylation play in its function?

ITCH (also known as AIP4, NAPP1, or E3 ubiquitin-protein ligase Itchy homolog) is a HECT-type E3 ubiquitin ligase that mediates ubiquitination of various protein substrates, targeting them for degradation. The phosphorylation at tyrosine 420 (Y420) represents a critical regulatory modification that modulates ITCH's substrate binding capacity and enzymatic activity.

Phosphorylation at Y420 is mediated primarily by the Fyn kinase, a member of the Src family kinases. This post-translational modification occurs within ITCH's protein-protein interaction domain and has been demonstrated to decrease ITCH's binding to certain substrates, including JUNB . This inhibitory phosphorylation serves as a regulatory mechanism that can dynamically control ITCH-mediated protein degradation pathways in response to cellular signaling events.

How does Phospho-ITCH (Y420) differ from other forms of phosphorylated ITCH?

ITCH can be phosphorylated at multiple residues by different kinases, each having distinct functional consequences:

The phospho-specific antibody against Y420 is designed to selectively recognize only ITCH molecules phosphorylated at this specific tyrosine residue, enabling researchers to specifically monitor this inhibitory modification without cross-reactivity to other phosphorylated forms . The antibody achieves this specificity through recognition of the unique amino acid sequence surrounding Y420, typically represented as F-I-Y(p)-G-N .

What are the optimal methods for validating Phospho-ITCH (Y420) antibody specificity in experimental settings?

Multiple complementary approaches should be employed to confirm antibody specificity:

• Phosphopeptide competition assays: Pre-incubate the antibody with the phosphorylated peptide immunogen (containing pY420) before application to samples. This should abolish signal detection. In parallel, pre-incubation with non-phosphorylated peptide should not affect signal, confirming phospho-specificity .

• Site-directed mutagenesis validation: Express wild-type ITCH alongside a Y420F mutant (tyrosine replaced with phenylalanine to prevent phosphorylation). The antibody should detect wild-type protein following Fyn activation but show no signal with the Y420F mutant .

• Phosphatase treatment: Treat sample duplicates with lambda phosphatase prior to analysis. This dephosphorylation should eliminate antibody recognition if it truly targets only the phosphorylated form .

• Kinase manipulation: Inhibition of Fyn kinase (e.g., using PP2 inhibitor) should reduce Phospho-ITCH (Y420) detection, while Fyn activation should increase signal intensity .

These validation steps collectively ensure that the observed signal genuinely represents phosphorylation at Y420 and not cross-reactivity with other epitopes or phosphorylation sites.

Western Blotting Protocol Optimization:

• Sample preparation: Lyse cells in buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktail) to preserve phosphorylation status.

• Dilution optimization: Most Phospho-ITCH (Y420) antibodies work optimally at dilutions between 1:500-1:1000 for Western blotting .

• Blocking recommendations: Use 5% BSA in TBST rather than milk, as milk contains casein phosphoproteins that can interfere with phospho-antibody binding.

• Membrane considerations: PVDF membranes typically provide better results than nitrocellulose for phospho-epitope detection.

Immunohistochemistry Conditions:

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 15-20 minutes optimizes phospho-epitope exposure in formalin-fixed tissues.

• Antibody dilution: Typically 1:100-1:300 for IHC applications .

• Detection system: Amplification systems (e.g., tyramide signal amplification) may be necessary for low-abundance phospho-proteins.

• Validation controls: Adjacent tissue sections should be stained with both phospho-specific and total ITCH antibodies to assess relative phosphorylation levels.

These optimized conditions help ensure specific and sensitive detection of the Phospho-ITCH (Y420) epitope while minimizing background and non-specific binding.

How can Phospho-ITCH (Y420) antibodies be used to investigate AMPK-dependent regulation of Notch1 stability?

Recent research has unveiled a mechanistic link between AMPK signaling, ITCH phosphorylation, and Notch1 stability in hypoxic conditions . To investigate this pathway:

• Experimental design approach:

  • Establish hypoxic and normoxic cell culture conditions (1-2% O₂ vs. 21% O₂)

  • Manipulate AMPK activity using compound C (inhibitor) or AICAR/metformin (activators)

  • Monitor both Phospho-ITCH (Y420) levels and Notch1 stability

• Methodological workflow:

  • Co-immunoprecipitation of ITCH followed by phosphotyrosine detection using anti-phosphotyrosine antibodies (4G10 or pY20)

  • Direct detection of Phospho-ITCH (Y420) by Western blotting

  • Assessment of ITCH-Notch1 interaction through reciprocal co-immunoprecipitation

  • Ubiquitination assays using K48-specific ubiquitin antibodies to measure Notch1 ubiquitination

• Mechanistic validation:

  • Express dominant-negative Fyn to disrupt the AMPK-Fyn-ITCH axis

  • Use phospho-mimetic (Y420E) or phospho-deficient (Y420F) ITCH mutants

  • Employ CRISPR/Cas9 gene editing to introduce these mutations at endogenous loci

This experimental approach allows for detailed characterization of how AMPK activation in hypoxia leads to Fyn-mediated phosphorylation of ITCH at Y420, thereby reducing ITCH-mediated ubiquitination of Notch1 and promoting Notch1 stability .

What approaches can be used to distinguish between total ITCH levels and Phospho-ITCH (Y420) in complex tissue samples?

Analyzing both total ITCH and its phosphorylated form in tissues requires careful methodological consideration:

• Sequential immunofluorescence staining:

  • First round: Use Phospho-ITCH (Y420) antibody with one fluorophore (e.g., Alexa Fluor 488)

  • Image acquisition

  • Antibody stripping using glycine buffer (pH 2.5)

  • Second round: Total ITCH antibody with different fluorophore (e.g., Alexa Fluor 594)

  • Calculate phosphorylation ratio by dividing phospho-signal by total signal in each cell/region

• Multiplex immunohistochemistry:

  • Employ tyramide signal amplification (TSA) system for sequential staining

  • Use multispectral imaging systems to separate signals

  • Perform automated image analysis for phospho/total quantification

• Laser capture microdissection and Western blotting:

  • Isolate regions of interest from tissue sections

  • Extract proteins and perform Western blotting for both phospho and total ITCH

  • Normalize phospho-signal to total protein level

• Proximity ligation assay (PLA):

  • Combine Phospho-ITCH (Y420) and total ITCH antibodies from different species

  • Use species-specific PLA probes to generate signal only when both antibodies are in close proximity

  • This approach specifically detects the phosphorylated subpopulation in situ

These approaches enable quantitative assessment of ITCH phosphorylation state in heterogeneous tissues while controlling for variations in total protein expression.

Sources of False Positives:

• Cross-reactivity with related phospho-tyrosine motifs:

  • The sequence surrounding Y420 (FIYGN) may share homology with other phospho-proteins

  • Solution: Perform peptide competition assays with both target and potential cross-reactive peptides

• Non-specific binding to denatured proteins:

  • Solution: Optimize blocking conditions using 5% BSA instead of milk; include 0.1% Tween-20 in antibody diluent

• Rapid spontaneous phosphorylation during sample preparation:

  • Solution: Use phosphatase inhibitors (10mM sodium fluoride, 1mM sodium orthovanadate) in all buffers from cell lysis onwards

Sources of False Negatives:

• Rapid dephosphorylation during sample preparation:

  • Solution: Maintain samples at 4°C throughout processing; add phosphatase inhibitor cocktails

• Epitope masking in fixed tissues:

  • Solution: Optimize antigen retrieval methods; test multiple pH conditions (pH 6.0 citrate buffer vs. pH 9.0 EDTA buffer)

• Low sensitivity of detection method:

  • Solution: Use enhanced chemiluminescence (ECL) substrates for Western blot; implement signal amplification steps for IHC/IF

• Competitive binding from endogenous phosphatases:

  • Solution: Pre-clear lysates with protein A/G beads before immunoprecipitation

Implementing these troubleshooting approaches can significantly improve the reliability and reproducibility of experiments utilizing Phospho-ITCH (Y420) antibodies.

How can researchers distinguish between specific Phospho-ITCH (Y420) signal and background in multi-parametric flow cytometry?

Flow cytometry with phospho-specific antibodies requires rigorous controls and optimization:

• Essential Controls Framework:

  • Fluorescence Minus One (FMO) controls to set proper compensation

  • Isotype controls matched to antibody class and conjugate

  • Biological controls: Fyn kinase inhibitor-treated (negative) and constitutively active Fyn-expressing cells (positive)

  • Phosphatase-treated samples as technical negative controls

• Signal Validation Approach:

  • Compare staining patterns between permeabilized and non-permeabilized cells

  • Implement Y420F mutant-expressing cells as gold-standard negative controls

  • Use phospho-flow compatible fixation (paraformaldehyde) and permeabilization (methanol or commercial permeabilization buffers)

• Optimization Parameters:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Test multiple permeabilization protocols (Triton X-100, saponin, methanol)

  • Implement sequential staining: surface markers first, followed by fixation, permeabilization, and phospho-staining

• Data Analysis Strategies:

  • Gate on singlets and viable cells before phospho-signal analysis

  • Use biexponential display for phospho-signals rather than logarithmic scales

  • Calculate phospho-protein Staining Index: (Median Positive - Median Negative)/2 × Standard Deviation of Negative

These approaches enable reliable discrimination between specific Phospho-ITCH (Y420) signal and background fluorescence in complex cell populations.

How might single-cell analysis techniques be adapted to study ITCH phosphorylation heterogeneity in tissue samples?

Single-cell analysis of phosphorylation states represents a frontier in phospho-protein research:

• Single-cell phospho-proteomics approaches:

  • Mass cytometry (CyTOF) using metal-conjugated Phospho-ITCH (Y420) antibodies

  • Integration with other cell type markers and signaling molecules

  • Implementation of dimensionality reduction algorithms (tSNE, UMAP) to identify cell populations with distinct phosphorylation profiles

• Spatial transcriptomics correlation:

  • Combine phospho-ITCH immunofluorescence with RNA-seq on the same tissue section

  • Correlate phosphorylation status with gene expression signatures

  • Identify molecular pathways activated in cells with high vs. low Y420 phosphorylation

• In situ proximity ligation adaptations:

  • Develop rolling circle amplification-based detection of phospho-ITCH

  • Combine with RNA fluorescence in situ hybridization (FISH) for multi-omic analysis

  • Implement automated image analysis algorithms for quantitative assessment

• Microfluidic single-cell Western blotting:

  • Adapt protocols for detecting phospho-ITCH in individual cells

  • Correlate with functional readouts like single-cell ubiquitination assays

These emerging technologies promise to reveal cell-to-cell variability in ITCH phosphorylation status that may be masked in bulk tissue analyses, potentially uncovering new regulatory mechanisms and cellular subpopulations.

What recent technological advances might improve detection sensitivity and specificity for Phospho-ITCH (Y420) in challenging samples?

Several cutting-edge approaches offer improved phospho-protein detection:

• Next-generation antibody engineering:

  • Single-chain variable fragments (scFvs) with enhanced phospho-specificity

  • Recombinant phospho-specific nanobodies with superior tissue penetration

  • Bi-specific antibodies targeting both ITCH protein backbone and Y420 phosphorylation site

• Alternative affinity reagents:

  • Synthetically evolved phospho-binding domains

  • DNA aptamers selected against Phospho-ITCH (Y420)

  • Molecularly imprinted polymers (MIPs) as artificial antibody substitutes

• Signal amplification technologies:

  • Hybridization chain reaction (HCR) amplification for immunohistochemistry

  • Proximity extension assays for ultra-sensitive detection in limited samples

  • Electrochemiluminescent detection systems with femtomolar sensitivity

• Computational approaches:

  • Deep learning algorithms for automated identification of true phospho-signals

  • Integration of multiple antibody validation datasets to assess reliability

  • Predictive modeling of phosphorylation dynamics based on kinase activity profiles

These technological advances could significantly enhance our ability to detect and quantify Phospho-ITCH (Y420) in challenging samples like archived tissues, minute biopsy specimens, or cells with low ITCH expression levels.

How does Phospho-ITCH (Y420) status correlate with hypoxia response mechanisms in cancer progression?

The relationship between ITCH phosphorylation and cancer hypoxia response involves several interconnected pathways:

• Molecular mechanism framework:

  • Hypoxia activates AMPK signaling pathways

  • AMPK regulates Fyn kinase activity through modulation of inhibitory phosphatases

  • Activated Fyn phosphorylates ITCH at Y420

  • Phosphorylated ITCH exhibits reduced binding to Notch1

  • Decreased Notch1 ubiquitination leads to Notch1 stabilization and increased signaling

• Experimental assessment approach:

  • Analyze patient tumor samples for correlation between hypoxic markers (HIF-1α, CA-IX), Phospho-ITCH (Y420), and Notch1 levels

  • Develop tissue microarray-based scoring system for Phospho-ITCH (Y420) in tumor hypoxic regions

  • Correlate Phospho-ITCH (Y420) levels with clinical outcomes and treatment resistance

• Functional validation strategy:

  • Generate cancer cell lines expressing phospho-deficient ITCH (Y420F)

  • Assess impact on tumor growth in hypoxic microenvironments

  • Evaluate sensitivity to hypoxia-activated prodrugs and Notch inhibitors

This research framework provides insight into how ITCH phosphorylation may serve as a molecular switch that promotes cancer cell adaptation to hypoxic conditions through Notch1 signaling, potentially identifying new therapeutic targets and biomarkers.

How can researchers experimentally determine the functional consequences of Phospho-ITCH (Y420) on its substrate specificity?

Understanding how Y420 phosphorylation alters ITCH substrate recognition requires multifaceted approaches:

• Global substrate profiling:

  • Compare ubiquitinomes of cells expressing wild-type ITCH versus phospho-mimetic (Y420E) or phospho-deficient (Y420F) mutants

  • Implement stable isotope labeling with amino acids in cell culture (SILAC) with tandem ubiquitin binding entities (TUBEs) enrichment

  • Validate candidates using in vitro ubiquitination assays with recombinant proteins

• Structural biology approaches:

  • Conduct hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes induced by Y420 phosphorylation

  • Develop co-crystallization of ITCH WW domains with substrate peptides in phosphorylated versus non-phosphorylated states

  • Implement molecular dynamics simulations to predict altered binding interfaces

• Protein-protein interaction analyses:

  • BioID proximity labeling with wild-type versus Y420F/Y420E ITCH as baits

  • Quantitative ITCH interactome analysis following Fyn activation/inhibition

  • Microscale thermophoresis to measure binding affinities to known substrates

• Domain-specific functions:

  • Generate chimeric proteins with individual domains from phosphorylated/non-phosphorylated ITCH

  • Assess domain-specific contributions to altered substrate recognition

  • Implement intramolecular FRET sensors to detect phosphorylation-induced conformational changes