Human USP18 (UBP43) knockout A549 cell line
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- WB
Lab
Western blot - Human USP18 (UBP43) knockout A549 cell line (AB267177)
False colour image of Western blot : Anti-USP18 antibody staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to USP18. A band was observed at 34/37 kDa in wild-type A549 cell lysates with no signal observed at this size in USP18 knockout cell line ab267177 (knockout cell lysate ab258751). To generate this image, wild-type and USP18 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes:
Anti-USP18 antibody at 1/1000 dilution
Lane 1:
Wild-type A549 Vehicle Control IFNa (0 IU/mL, 12 h) cell lysate at 20 µg
Lane 2:
Wild-type A549 Treated IFNa (100 IU/mL, 12 h) cell lysate at 20 µg
Lane 3:
USP18 knockout A549 Vehicle Control IFNa (0 IU/mL, 12 h) ab262530 cell lysate at 20 µg
Lane 4:
USP18 knockout A549 Treated IFNa (100 IU/mL, 12 h) ab262530 cell lysate at 20 µg
Secondary
Lanes 1 - 4:
Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution
Lanes 1 - 4:
Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Predicted band size: 43 kDa
Observed band size: 34-37 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human USP18 (UBP43) knockout A549 cell line (AB267177)
Allele-3 : 1 bp deletion in exon 4.
- Sanger seq
Unknown
Sanger Sequencing - Human USP18 (UBP43) knockout A549 cell line (AB267177)
Allele-1 : 17 bp deletion in exon4
- Sanger seq
Unknown
Sanger Sequencing - Human USP18 (UBP43) knockout A549 cell line (AB267177)
Allele-2 : 7 bp deletion in exon 4.
- ICC/IF
Supplier Data
Immunocytochemistry/ Immunofluorescence - Human USP18 (UBP43) knockout A549 cell line (AB267177)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Parental A549 (human lung carcinoma epithelial cell); USP18 KO A549 (USP18 knockout human lung carcinoma epithelial cell) (ab267177); USP18 KO A549 treated with human IFNa (100 U/mL) for 14 hr cells labelling UBP43/USP18 with ab325178 at 1/100 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).
Confocal image showing cytoplasmic and nuclear staining in Parental A549 cells (shown in green) treated with human IFNa (100 U/mL) for 14 hr. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.
Complementary Products
Product details
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant 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
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.
- Do not allow the cell density to exceed 7x104 cells/cm2.
Culture medium
F-12K + 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
UBP43 regulates type I interferon signaling and affects immune response. As a negative feedback regulator it inhibits the interferon signaling pathway preventing unnecessary immune reactions. UBP43 does not form a part of a larger protein complex but acts independently to maintain cellular homeostasis. Its activity impacts the function of other proteins involved in response to viral infections.
Pathways
UBP43 plays an important role in the JAK-STAT signaling pathway interacting with proteins involved in cytokine signaling. It influences the pathway by inhibiting the action of interferon-stimulated genes therefore modulating the immune response. UBP43's interaction with ISG15 a ubiquitin-like modifier is also a critical aspect that affects other cellular processes like protein modification and response to inflammatory stimuli.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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