The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/10000. Detects a band of approximately 103 kDa (predicted molecular weight: 103 kDa).
Is unsuitable for Flow Cyt,ICC,IHC-P or IP.
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.
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.
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.
Cell membrane. Cytoplasm. Nucleus. Associates with endocytic vesicles. May be recruited to exosomes by NDFIP1.
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
NFE2 associated polypeptide 1 antibody
NFE2-associated polypeptide 1 antibody
Ubiquitin protein ligase ITCH antibody
Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
Predicted band size : 103 kDa Additional bands at : 110 kDa. We are unsure as to the identity of these extra bands.
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 - 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