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    products/cell-lines/human-itch-aip4-knockout-hela-cell-line-ab265338.pdf

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Cell Biology Proteolysis / Ubiquitin Proteasome / Ubiquitin Ubiquitin E3 Enzymes Hect E3 Ligase
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Human ITCH (AIP4) knockout HeLa cell line (ab265338)

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Western blot - Human ITCH knockout HeLa cell line (ab265338)
  • Sanger Sequencing - Human ITCH knockout HeLa cell line (ab265338)

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Overview

  • Product name

    Human ITCH (AIP4) knockout HeLa cell line
  • Parental Cell Line

    HeLa
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 7
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing, Western Blot (WB)
  • Tested applications

    Suitable for: WBmore details
  • Biosafety level

    2
  • General notes

    Recommended control: Human wild-type HeLa cell line (ab255928). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.

    Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

    Culture medium: DMEM (High Glucose) + 10% FBS

    Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.

    1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
    2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
    3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
    4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.

    Subculture guidelines:

    • All seeding densities should be based on cell counts gained by established methods.
    • A guide seeding density of 2x104 cells/cm2 is recommended.
    • A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
    • Cells should be passaged when they have achieved 80-90% confluence.

    This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

    We will provide viable cells that proliferate on revival.

Properties

  • Number of cells

    1 x 106 cells/vial, 1 mL
  • Adherent /Suspension

    Adherent
  • Tissue

    Cervix
  • Cell type

    epithelial
  • Disease

    Adenocarcinoma
  • Gender

    Female
  • STR Analysis

    Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10
  • Mycoplasma free

    Yes
  • Storage instructions

    Shipped on Dry Ice. Store in liquid nitrogen.
  • Storage buffer

    Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
  • Research areas

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

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

Associated products

  • KO cell lysates

    • Human ITCH (AIP4) knockout HeLa cell lysate (ab258014)
  • Related Products

    • Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)
    • Anti-ITCH/AIP4 antibody [EPR4936] - BSA and Azide free (ab247678)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab265338 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
Use at an assay dependent concentration. Predicted molecular weight: 103 kDa.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 103 kDa.

Images

  • Western blot - Human ITCH knockout HeLa cell line (ab265338)
    Western blot - Human ITCH knockout HeLa cell line (ab265338)
    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.

  • Sanger Sequencing - Human ITCH knockout HeLa cell line (ab265338)
    Sanger Sequencing - Human ITCH knockout HeLa cell line (ab265338)
    Homozygous: 1 bp insertion in exon 7.

Protocols

  • Hemocytometer protocol
  • Mammalian cell tissue culture techniques protocol

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab265338? Please let us know so that we can cite the reference in this datasheet.

ab265338 has not yet been referenced specifically in any publications.

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