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Cell Biology Proteolysis / Ubiquitin Proteasome / Ubiquitin Ubiquitin E3 Enzymes Hect E3 Ligase
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)

  • Datasheet
  • SDS
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Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
  • Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
  • Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
  • Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
  • Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR4936] to ITCH/AIP4
  • Suitable for: WB
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Carrier Free

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Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
Knockout
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Human ITCH (AIP4) knockout HeLa cell line (ab265338)
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Anti-CBL antibody [YE323] - C-terminal (ab32027)

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Overview

  • Product name

    Anti-ITCH/AIP4 antibody [EPR4936]
    See all ITCH/AIP4 primary antibodies
  • Description

    Rabbit monoclonal [EPR4936] to ITCH/AIP4
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WBmore details
    Unsuitable for: Flow Cyt,ICC/IF,IHC-P or IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: K562, HeLa, HAP1 and 293T cell lysates. Rat and Mouse brain tissue lysates.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at -20°C. Stable for 12 months at -20°C.
  • Storage buffer

    pH: 7.20
    Preservative: 0.05% Sodium azide
    Constituents: 0.1% BSA, 40% Glycerol (glycerin, glycerine), 9.85% Tris glycine, 50% Tissue culture supernatant
  • Concentration information loading...
  • Purity

    Tissue culture supernatant
  • Clonality

    Monoclonal
  • Clone number

    EPR4936
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Proteolysis / Ubiquitin
    • Proteasome / Ubiquitin
    • Ubiquitin E3 Enzymes
    • Hect E3 Ligase

Associated products

  • Alternative Versions

    • Anti-ITCH/AIP4 antibody [EPR4936] - BSA and Azide free (ab247678)
  • Compatible Secondaries

    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human ITCH (AIP4) knockout HeLa cell line (ab265338)
  • KO cell lysates

    • Human ITCH (AIP4) knockout HeLa cell lysate (ab258014)
  • Positive Controls

    • K-562 nuclear extract lysate (ab14851)
    • HeLa membrane extract lysate (ab29547)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab108515 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB
1/1000 - 1/10000. Detects a band of approximately 103 kDa (predicted molecular weight: 103 kDa).
Notes
WB
1/1000 - 1/10000. Detects a band of approximately 103 kDa (predicted molecular weight: 103 kDa).
Application notes
Is unsuitable for Flow Cyt,ICC/IF,IHC-P or IP.

Target

  • Function

    Acts as an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. It catalyzes 'Lys-29'-, 'Lys-48'- and 'Lys-63'-linked ubiquitin conjugation. It is involved in the control of inflammatory signaling pathways. Is an essential component of a ubiquitin-editing protein complex, comprising also TNFAIP3, TAX1BP1 and RNF11, that ensures the transient nature of inflammatory signaling pathways. Promotes the association of the complex after TNF stimulation. Once the complex is formed, TNFAIP3 deubiquitinates 'Lys-63' polyubiquitin chains on RIPK1 and catalyzes the formation of 'Lys-48'-polyubiquitin chains. This leads to RIPK1 proteosomal degradation and consequently termination of the TNF- or LPS-mediated activation of NFKB1. Ubiquitinates RIPK2 by 'Lys-63'-linked conjugation and influences NOD2-dependent signal transduction pathways. Regulates the transcriptional activity of several transcription factors, and probably plays an important role in the regulation of immune response. Ubiquitinates NFE2 by 'Lys-63' linkages and is implicated in the control of the development of hematopoietic lineages. Critical regulator of T helper (TH2) cytokine development through its ability to induce JUNB ubiquitination and degradation (By similarity). Ubiquitinates SNX9. Ubiquitinates CXCR4 and HGS/HRS and regulates sorting of CXCR4 to the degradative pathway. It is involved in the negative regulation of MAVS-dependent cellular antiviral responses. Ubiquitinates MAVS through 'Lys-48'-linked conjugation resulting in MAVS proteosomal degradation. Involved in the regulation of apoptosis and reactive oxygen species levels through the ubiquitination and proteosomal degradation of TXNIP. Mediates the antiapoptotic activity of epidermal growth factor through the ubiquitination and proteosomal degradation of p15 BID. Targets DTX1 for lysosomal degradation and controls NOTCH1 degradation, in the absence of ligand, through 'Lys-29'-linked polyubiquitination.
  • Tissue specificity

    Widely expressed.
  • Pathway

    Protein modification; protein ubiquitination.
  • Involvement in disease

    Defects in ITCH are the cause of syndromic multisystem autoimmune disease (SMAD) [MIM:613385]. SMAD is characterized by organomegaly, failure to thrive, developmental delay, dysmorphic features and autoimmune inflammatory cell infiltration of the lungs, liver and gut.
  • Sequence similarities

    Contains 1 C2 domain.
    Contains 1 HECT (E6AP-type E3 ubiquitin-protein ligase) domain.
    Contains 4 WW domains.
  • Post-translational
    modifications

    On T-cell activation, phosphorylation by the JNK cascade on serine and threonine residues surrounding the PRR domain accelerates the ubiquitination and degradation of JUN and JUNB. The increased ITCH catalytic activity due to phosphorylation by JNK1 may occur due to a conformational change disrupting the interaction between the PRR/WW motifs domain and the HECT domain and, thus exposing the HECT domain (By similarity). Phosphorylation by FYN reduces interaction with JUNB and negatively controls JUN ubiquitination and degradation.
    Ubiquitinated; autopolyubiquitination with 'Lys-63' linkages which does not lead to protein degradation.
  • Cellular localization

    Cell membrane. Cytoplasm. Nucleus. Associates with endocytic vesicles. May be recruited to exosomes by NDFIP1.
  • Target information above from: UniProt accession Q96J02 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 83737 Human
    • Entrez Gene: 16396 Mouse
    • Entrez Gene: 311567 Rat
    • Omim: 606409 Human
    • SwissProt: Q96J02 Human
    • SwissProt: Q8C863 Mouse
    • Unigene: 632272 Human
    • Alternative names

      • ADMFD antibody
      • AIF4 antibody
      • AIP4 antibody
      • Atrophin 1 interacting protein 4 antibody
      • Atrophin-1-interacting protein 4 antibody
      • dJ468O1.1 antibody
      • dJ468O1.1 (atrophin 1 interacting protein 4 (AIP4)) antibody
      • dJ468O1.1 atrophin 1 interacting protein 4 AIP4 antibody
      • E3 ubiquitin protein ligase Itchy homolog antibody
      • E3 ubiquitin-protein ligase Itchy homolog antibody
      • EC 6.3.2 antibody
      • Itch antibody
      • ITCH_HUMAN antibody
      • Itchy E3 ubiquitin protein ligase antibody
      • Itchy E3 ubiquitin protein ligase homolog antibody
      • Itchy E3 ubiquitin protein ligase homolog mouse antibody
      • Itchy E3 ubiquitin protein ligase, mouse, homolog of antibody
      • Itchy homolog E3 ubiquitin protein ligase antibody
      • Itchy mouse homolog E3 ubiquitin protein ligase antibody
      • NAPP1 antibody
      • NFE2 associated polypeptide 1 antibody
      • NFE2-associated polypeptide 1 antibody
      • Ubiquitin protein ligase ITCH antibody
      see all

    Images

    • Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
      Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
      All lanes : Anti-ITCH/AIP4 antibody [EPR4936] (ab108515) at 1/1000 dilution

      Lane 1 : Wild-type HeLa cell lysate
      Lane 2 : ITCH knockout HeLa cell lysate
      Lane 3 : HAP1 cell lysate
      Lane 4 : K-562 cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

      Predicted band size: 103 kDa
      Observed band size: 103 kDa



      Lanes 1-4: Merged signal (red and green). Green - ab108515 observed at 103 kDa. Red - loading control ab8245 observed at 36 kDa. 

       ab108515 Anti-ITCH/AIP4 antibody [EPR4936] was shown to specifically react with ITCH/AIP4 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265338 (knockout cell lysate ab258014) was used.  Wild-type and ITCH/AIP4 knockout samples were subjected to SDS-PAGE.  ab108515 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively.  Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

    • Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
      Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)

      Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: ITCH/AIP4 knockout HAP1 cell lysate (20 µg)
      Lane 3: HeLa cell lysate (20 µg)
      Lane 4: K562 cell lysate (20 µg)
      Lanes 1 - 4: Merged signal (red and green). Green - ab108515 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab108515 was shown to specifically react with ITCH/AIP4 when ITCH/AIP4 knockout samples were used. Wild-type and ITCH/AIP4 knockout samples were subjected to SDS-PAGE. ab108515 and ab8245 (loading control to GAPDH) were diluted 1/500 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

    • Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
      Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
      All lanes : Anti-ITCH/AIP4 antibody [EPR4936] (ab108515) at 1/1000 dilution

      Lane 1 : K562 cell lysate
      Lane 2 : HeLa cell lysate
      Lane 3 : 293T cell lysate

      Lysates/proteins at 10 µg per lane.

      Predicted band size: 103 kDa

    • Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
      Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
      All lanes : Anti-ITCH/AIP4 antibody [EPR4936] (ab108515) at 1/1000 dilution

      Lane 1 : Mouse brain lysate
      Lane 2 : Rat brain lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 103 kDa
      Observed band size: 103 kDa


      Exposure time: 1 minute


      Blocking buffer and concentration: 5% NFDM/TBST

    • Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
      Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)

    Protocols

    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (2)

    Publishing research using ab108515? Please let us know so that we can cite the reference in this datasheet.

    ab108515 has been referenced in 2 publications.

    • Abe Y  et al. MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex. Commun Biol 2:23 (2019). PubMed: 30675521
    • Xu J  et al. NEDD4 protects vascular endothelial cells against Angiotensin II-induced cell death via enhancement of XPO1-mediated nuclear export. Exp Cell Res 383:111505 (2019). PubMed: 31326389

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