Human ITCH (AIP4) knockout HeLa cell line
- Advanced Validation
- What is this?
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ITCH KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 7. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
View Alternative Names
ADMFD, AIF4, Aip-4, Atrophin-1-interacting protein 4, E3 ubiquitin-protein ligase Itchy homolog, EC 6.3.2, ITCH_HUMAN, Itchy E3 ubiquitin protein ligase, Itchy E3 ubiquitin protein ligase homolog, Itchy E3 ubiquitin protein ligase homolog mouse, Itchy E3 ubiquitin protein ligase, mouse, homolog of, Itchy homolog E3 ubiquitin protein ligase, Itchy mouse homolog E3 ubiquitin protein ligase, NAPP1, NFE2-associated polypeptide 1, Ubiquitin protein ligase ITCH, dJ468O1.1, dJ468O1.1 (atrophin 1 interacting protein 4 (AIP4)), dJ468O1.1 atrophin 1 interacting protein 4 AIP4
- WB
Lab
Western blot - Human ITCH (AIP4) knockout HeLa cell line (AB265338)
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° 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.
All lanes:
Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (<a href='/en-us/products/primary-antibodies/itch-aip4-antibody-epr4936-ab108515'>ab108515</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
ITCH knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human ITCH (AIP4) knockout HeLa cell line (ab265338)
Lane 3:
HAP1 cell lysate at 20 µg
Lane 4:
K-562 cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution
Predicted band size: 103 kDa,43 kDa
Observed band size: 103 kDa,57 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human ITCH (AIP4) knockout HeLa cell line (AB265338)
Homozygous : 1 bp insertion in exon 7.
Reactivity data
Product details
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
Handling procedures
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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate 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. 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.
- Cells should be passaged when they have achieved 80-90% confluence.
Culture medium
DMEM (High Glucose) + 10% FBS
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
ITCH/AIP4 is integral in cellular regulatory mechanisms. It forms part of a sophisticated complex involved in immune response modulation cell cycle regulation and apoptosis. ITCH influences T-cell receptor signaling by controlling the degradation of proteins involved in these critical pathways. Through the ubiquitination cascade ITCH maintains cellular homeostasis participating in the intricate balance of protein synthesis and degradation necessary for normal cellular function.
Pathways
ITCH/AIP4 plays essential roles in the T-cell receptor and Wnt signaling pathways. It interacts with other proteins such as NEDD4 and CBL which are also E3 ubiquitin ligases to modulate signaling cascades involved in immune responses and cellular growth. In the Wnt pathway ITCH regulates the stability of Dishevelled proteins thereby affecting cellular proliferation and differentiation processes. This involvement in multiple pathways highlights its regulatory versatility and importance in maintaining cellular balance.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Complementary Products
![Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (AB108515)](https://content.abcam.com/products/images/itch-aip4-antibody-epr4936-ab108515--western-blot-img2865.jpg)
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com