Phospho-ITCH (Tyr420) Antibody

What is ITCH protein and why is Tyr420 phosphorylation significant?

ITCH (also known as AIP4) is a member of the NEDD4-like protein family of E3 ubiquitin-ligase molecules that regulate key trafficking decisions, including targeting proteins to proteosomes or lysosomes. The protein contains four tandem WW domains and a HECT domain, acting as a transcriptional corepressor of p45/NFE2 and participating in immune response regulation by modifying Notch-mediated signaling .

Tyrosine phosphorylation at position 420 (Tyr420) represents a critical regulatory mechanism that negatively modulates ITCH activity toward its substrates . This phosphorylation occurs within the sequence context F-I-Y(p)-G-N and serves as a molecular switch affecting ITCH's ability to interact with and ubiquitinate target proteins .

What applications are suitable for Phospho-ITCH (Tyr420) antibodies?

Phospho-ITCH (Tyr420) antibodies have been validated for multiple applications:

When designing experiments, optimal dilutions should be determined empirically for each specific application and experimental system .

How should I properly store and handle Phospho-ITCH (Tyr420) antibodies?

For maximum stability and performance:

• Store antibodies at -20°C for long-term storage

• For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles

• Aliquot into single-use vials

• Most preparations are supplied in PBS with 50% glycerol and 0.02% sodium azide (pH 7.4)

• A slight precipitate may occasionally form and can be dissolved by gentle vortexing without affecting antibody performance

How is specificity of Phospho-ITCH (Tyr420) antibodies ensured?

Commercial Phospho-ITCH (Tyr420) antibodies undergo rigorous validation:

• Production against synthesized phosphopeptides derived from human ITCH around the phosphorylation site (F-I-Y(p)-G-N)

• Purification via affinity chromatography using epitope-specific immunogens

• Removal of non-phospho specific antibodies through chromatography using non-phosphopeptides

• Validation through blocking peptide experiments, where parallel samples are treated with the specific phosphopeptide to confirm binding specificity

What role does ITCH Tyr420 phosphorylation play in hypoxia and cancer signaling?

Research has revealed a complex signaling network involving ITCH phosphorylation under hypoxic conditions:

• Hypoxia induces increased tyrosine phosphorylation of immunoprecipitated ITCH

• AMPK mediates Itch phosphorylation in hypoxia, despite being a Ser/Thr kinase

• AMPK appears to act through Fyn, a Src family tyrosine kinase known to phosphorylate and inhibit ITCH activity at Tyr420

• This phosphorylation inhibits ITCH's ubiquitin ligase activity toward Notch1, stabilizing cleaved Notch1 levels

• Treatment with PP2 (a Src family kinase inhibitor) or expression of dominant-negative Fyn impairs hypoxia-triggered elevation in cleaved Notch1 levels

• Patient-derived breast cancer cells show elevated levels of phospho-ITCH under hypoxic conditions

These findings establish a critical AMPK-Fyn-ITCH-Notch1 signaling axis that appears relevant in breast cancer, with potential therapeutic implications.

What experimental controls should be included when using Phospho-ITCH (Tyr420) antibodies?

For rigorous experimental design with phospho-specific antibodies:

• Positive control: Include samples with known ITCH Tyr420 phosphorylation

  • Cells treated with hypoxia, which increases ITCH Tyr420 phosphorylation

  • Mitotic cell extracts, which contain active kinases capable of phosphorylating ITCH

• Negative controls:

  • Samples treated with phosphatase to remove phosphate groups

  • Cells expressing dominant-negative Fyn construct, which reduces Tyr-phosphorylation of ITCH

  • Cells with AMPK knockdown, which decreases ITCH Tyr420 phosphorylation

• Specificity controls:

  • Blocking peptide competition assays using the phosphopeptide immunogen

  • Parallel detection with a total ITCH antibody to normalize phospho-signal

• Assay controls:

  • For IHC: Include secondary-only controls and isotype controls

  • For western blots: Include molecular weight markers and loading controls

How can researchers generate phosphorylated ITCH protein for experimental purposes?

Several approaches for generating phosphorylated ITCH protein have been documented:

• In vitro enzymatic phosphorylation:

  • Express poly-His-tagged full-length ITCH in E. coli

  • Purify on Ni-NTA beads

  • Incubate bound protein with mitotic cell extract containing active kinases

  • Wash six times to remove unbound components

  • Elute with imidazole-containing buffer

  • Confirm phosphorylation via mobility shift assay

• Cell-based methods:

  • Subject cells to hypoxic conditions (typically 1% O₂)

  • Immunoprecipitate ITCH and confirm phosphorylation by immunoblotting with phospho-specific antibodies

  • Treatment with PP2 (Src family kinase inhibitor) can block this phosphorylation

• Modulation approaches:

  • Knockdown AMPK with doxycycline-inducible systems to reduce ITCH phosphorylation

  • Express dominant-negative Fyn constructs to inhibit phosphorylation

  • Treat with pharmacological AMPK inhibitors to reduce phospho-ITCH levels in hypoxic conditions

How can I incorporate Phospho-ITCH (Tyr420) antibodies into multi-parameter analyses?

For advanced experimental designs:

• Phospho-specific antibody sequencing (Phospho-seq):

  • Conjugate Phospho-ITCH (Tyr420) antibodies with DNA oligos containing unique barcode sequences

  • This allows multiplexing with other phospho-specific and non-phospho antibodies

  • Up to 100 antibodies can be used simultaneously in a single experiment

  • Compatible with fixed, permeabilized cells similar to intracellular flow cytometry

• Considerations for antibody selection in multi-parameter analyses:

  • Primary criteria: functionality in Intracellular Flow Cytometry (ICFC) or Immunocytochemistry (ICC)

  • Antibody sensitivity and background staining significantly impact results

  • Not all ICFC/ICC-validated antibodies work well with Phospho-seq due to fixation/permeabilization differences

• Compatible technology platforms:

  • Can be used with TSB tags (10X feature barcodes) or TSA tags (Poly A)

  • Capture with InCite-seq, NEAT-seq, or using Bridge Oligo A

  • Can be integrated with 10x Multiome protocols, though fixation affects RNA data quality

What is the relationship between ITCH phosphorylation and disease pathology?

Research indicates significant connections between ITCH phosphorylation and disease states:

• Cancer implications:

  • In breast cancer patient samples, there is an association between pACC (AMPK activity marker) and NICD (Notch intracellular domain) levels (p=0.0422, Fischer exact test)

  • Primary tissue-derived breast cancer cells show elevated phospho-ITCH levels under hypoxia

  • Pharmacological inhibition of AMPK reduces cleaved Notch1 and phospho-ITCH levels in these cells

• Gene signature correlations:

  • Analysis of TCGA primary breast cancer dataset shows correlation between AMPK, Hypoxia, and Notch pathway signatures

• Neurological disorders:

  • ITCH interacts with atrophin-1, which contains a polyglutamine repeat expansion responsible for dentatorubral and pallidoluysian atrophy

  • This suggests potential involvement in neurodegenerative disorders

• Immune system regulation:

  • ITCH participates in immune response regulation by modifying Notch-mediated signaling

  • Mouse Itch protein is implicated in the regulation and differentiation of erythroid and lymphoid cells

What are common issues when working with Phospho-ITCH (Tyr420) antibodies and how can they be resolved?

How can researchers verify that their antibody specifically recognizes phosphorylated versus non-phosphorylated ITCH?

To confirm phospho-specificity:

• Parallel Western blot analysis:

  • Run identical samples on two blots

  • Probe one with Phospho-ITCH (Tyr420) antibody

  • Probe the second with total ITCH antibody

  • Compare band patterns and intensities

• Phosphatase treatment control:

  • Split your sample in two

  • Treat one portion with lambda phosphatase

  • The signal should disappear in the phosphatase-treated sample

• Blocking peptide competition:

  • Pre-incubate antibody with phosphopeptide immunogen

  • Pre-incubate with non-phosphorylated peptide in parallel

  • Only the phosphopeptide should block specific binding

• Stimulation experiments:

  • Compare samples from cells under normal conditions versus hypoxia (which increases Tyr420 phosphorylation)

  • Compare cells with AMPK knockdown versus control cells

What considerations are important when selecting between different commercial Phospho-ITCH (Tyr420) antibodies?

When evaluating commercial antibodies:

• Validation method depth:

  • Look for antibodies validated using multiple techniques (Western blot, IHC, ELISA)

  • Check if phospho-specific blocking experiments were performed

  • Evaluate whether non-phospho antibody removal techniques were applied

• Immunogen design:

  • Confirm the immunogen is the specific phosphopeptide sequence (F-I-Y(p)-G-N)

  • Check the extent of surrounding sequence included in the immunogen

  • Evaluate host species and production method

• Cross-reactivity profile:

  • Verify species reactivity (human, mouse, etc.)

  • Check for potential cross-reactivity with related proteins

  • Review any provided cross-reactivity data

• Application-specific performance:

  • Some antibodies may perform better in certain applications

  • Review application-specific validation data

  • Consider recommended dilutions for your specific application