Human STAT1 knockout HeLa cell line (ab255346)
Overview
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Product name
Human STAT1 knockout HeLa cell line
See all STAT1 lysates -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 14 bp deletion in exon 4 and 1 bp deletion in exon 4 and 5 bp deletion in exon 4 -
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 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.
We will provide viable cells that proliferate on revival.
Properties
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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
Target
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Function
Signal transducer and activator of transcription that mediates signaling by interferons (IFNs). Following type I IFN (IFN-alpha and IFN-beta) binding to cell surface receptors, Jak kinases (TYK2 and JAK1) are activated, leading to tyrosine phosphorylation of STAT1 and STAT2. The phosphorylated STATs dimerize, associate with ISGF3G/IRF-9 to form a complex termed ISGF3 transcription factor, that enters the nucleus. ISGF3 binds to the IFN stimulated response element (ISRE) to activate the transcription of interferon stimulated genes, which drive the cell in an antiviral state. In response to type II IFN (IFN-gamma), STAT1 is tyrosine- and serine-phosphorylated. It then forms a homodimer termed IFN-gamma-activated factor (GAF), migrates into the nucleus and binds to the IFN gamma activated sequence (GAS) to drive the expression of the target genes, inducing a cellular antiviral state. -
Involvement in disease
Note=STAT1 deficiency results in impaired immune response leading to severe mycobacterial and viral diseases. In the case of complete deficiency, patients can die of viral disease.
Defects in STAT1 are a cause of mendelian susceptibility to mycobacterial disease (MSMD) [MIM:209950]; also known as familial disseminated atypical mycobacterial infection. This rare condition confers predisposition to illness caused by moderately virulent mycobacterial species, such as Bacillus Calmette-Guerin (BCG) vaccine and environmental non-tuberculous mycobacteria, and by the more virulent Mycobacterium tuberculosis. Other microorganisms rarely cause severe clinical disease in individuals with susceptibility to mycobacterial infections, with the exception of Salmonella which infects less than 50% of these individuals. The pathogenic mechanism underlying MSMD is the impairment of interferon-gamma mediated immunity whose severity determines the clinical outcome. Some patients die of overwhelming mycobacterial disease with lepromatous-like lesions in early childhood, whereas others develop, later in life, disseminated but curable infections with tuberculoid granulomas. MSMD is a genetically heterogeneous disease with autosomal recessive, autosomal dominant or X-linked inheritance. -
Sequence similarities
Belongs to the transcription factor STAT family.
Contains 1 SH2 domain. -
Post-translational
modificationsPhosphorylated on tyrosine and serine residues in response to IFN-alpha, IFN-gamma, PDGF and EGF. Phosphorylation on Tyr-701 (lacking in beta form) by JAK promotes dimerization and subsequent translocation to the nucleus. Phosphorylation on Ser-727 by several kinases including MAPK14, ERK1/2 and CAMKII on IFN-gamma stimulation, regulates STAT1 transcriptional activity. Phosphorylation on Ser-727 promotes sumoylation though increasing interaction with PIAS. Phosphorylation on Ser-727 by PKCdelta induces apoptosis in response to DNA-damaging agents.
Sumoylated by SUMO1, SUMO2 and SUMO3. Sumoylation is enhanced by IFN-gamma-induced phosphorylation on Ser-727, and by interaction with PIAS proteins. Enhances the transactivation activity.
ISGylated. -
Cellular localization
Cytoplasm. Nucleus. Translocated into the nucleus in response to IFN-gamma-induced tyrosine phosphorylation and dimerization. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab255346 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 |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 87 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 87 kDa. |
Images
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All lanes : Anti-STAT1 alpha antibody [EPYR2154] (ab92506) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : STAT1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 87 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?Lanes 1 - 2: Merged signal (red and green). Green - ab92506 observed at 85 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
ab92506 was shown to react with STAT1 alpha in wild-type HeLa cells in Western blot with loss of signal observed in STAT1 knockout cell line ab255346 (STAT1 knockout cell lysate ab263837). Wild-type HeLa and STAT1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab92506 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
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All lanes : Anti-STAT1 antibody [EPR4407] (ab109320) at 1/10000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : STAT1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 87 kDa
Observed band size: 87 kDaLanes 1- 2: Merged signal (red and green). Green - ab109320 observed at 87 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab109320 was shown to react with STAT1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255346 (knockout cell lysate ab263837) was used. Wild-type HeLa and STAT1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109320 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 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.
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Allele-1: 14 bp deletion in exon 4.
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Allele-2: 5 bp deletion in exon 4.
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Allele-3: 1 bp deletion in exon 4.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab255346 has not yet been referenced specifically in any publications.