Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line
- Advanced Validation
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IRF9 KO cell line available to order. KO validated by Immunocytochemistry. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and 2 bp deletion in exon 2. To order both knockout and wild-type control cells: select 2 x 1000000Cells/vial. To order only knockout cells: select 1000000Cells/vial.
View Alternative Names
IFN-alpha-responsive transcription factor subunit, IRF9_HUMAN, ISGF-3, ISGF-3 gamma, ISGF3 p48 subunit, ISGF3G, Interferon regulatory factor 9, Interferon-stimulated gene factor 3 gamma, OTTHUMP00000164692, OTTHUMP00000164693, P48, Transcriptional regulator ISGF3 subunit gamma, interferon stimulated transcription factor 3, interferon-stimulated transcription factor 3, gamma 48kDa
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (AB266051)
ab271043 staining Interferon regulatory factor 9 in wild-type HeLa cells and IRF9 knockout HeLa cells treated with interferon-a1 (ab48750) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab271043 at 0.4 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
- WB
Lab
Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (AB266051)
False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] staining at 2 μg/ml shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution shown in red. In Western blot ab277803 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in IRF9 CRISPR-Cas9 edited cell line ab266051 (CRISPR-Cas9 edited cell lysate ab258472). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image wild-type and IRF9 CRISPR-Cas9 edited HeLa 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 °. 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
All lanes:
Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] (<a href='/en-us/products/primary-antibodies/interferon-regulatory-factor-9-irf-9-antibody-14hclc-ab277803'>ab277803</a>) at 2 µg/mL
Lane 1:
Wild-type HeLa treated hIFN-a1 (10 ng/ml, 16 h) cell lysate at 20 µg
Lane 2:
IRF9 knockout HeLa treated hIFN-a1 (10 ng/ml, 16 h) cell lysate at 20 µg
Lane 2:
Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (ab266051)
Lane 3:
Wild-type HeLa vehicle control hIFN-a1 (0 ng/ml, 16 h) cell lysate at 20 µg
Lane 4:
IRF9 knockout HeLa vehicle control hIFN-a1 (0 ng/ml, 16 h) cell lysate at 20 µg
Predicted band size: 44 kDa
Observed band size: 48 kDa
false
- WB
Lab
Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (AB266051)
False colour image of Western blot : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] staining at 1/1000 dilution shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution shown in red. In Western blot ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in IRF9 CRISPR-Cas9 edited cell line ab266051 (CRISPR-Cas9 edited cell lysate ab258472). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image wild-type and IRF9 CRISPR-Cas9 edited HeLa cell lysates were analysed. First samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4°. 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
All lanes:
Western blot - Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (<a href='/en-us/products/primary-antibodies/interferon-regulatory-factor-9-irf-9-antibody-epr24260-55-ab271043'>ab271043</a>) at 1/1000 dilution
Lane 1:
wild-type HeLa Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate at 20 µg
Lane 2:
IRF9 knockout HeLa treated: hIFN-a1 (10 ng/ml, 16 h) cell lysate at 20 µg
Lane 2:
Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (ab266051)
Lane 3:
wild-type HeLa Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate at 20 µg
Lane 4:
IRF9 knockout HeLa vehicle control: hIFN-a1 (0 ng/ml, 16 h) cell lysate at 20 µg
Lane 5:
THP-1 cell lysate at 20 µg
Lane 6:
ACHN cell lysate at 20 µg
Predicted band size: 44 kDa
Observed band size: 48 kDa
false
- WB
Lab
Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (AB266051)
Lanes 1 - 4 : Merged signal (red and green). Green - Anti-IRF9 antibody observed at 48 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa. Anti-IRF9 antibody was shown to react with IRF-9 in wild-type HeLa cells in western blot. The bands observed in IRF9 knockout cell line ab266051 (IRF9 knockout cell lysate ab258472) below 48 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. HeLa wild-type and IRF9 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with anti-IRF9 antibody and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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.
All lanes:
Anti-IRF9 antibody at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
IRF9 knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (ab266051)
Lane 3:
THP-1 cell lysate at 20 µg
Lane 4:
ACHN cell lysate at 20 µg
false
- Sanger seq
Unknown
Sanger Sequencing - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (AB266051)
Allele-1 : 1 bp insertion in exon2
- Sanger seq
Unknown
Sanger Sequencing - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (AB266051)
Allele-2 : 2 bp deletion in exon 2.
Reactivity data
Product details
We will provide viable cells that proliferate on revival.
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.
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
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
IRF-9 participates in the assembly of the transcription factor complex ISGF3 alongside STAT1 and STAT2. This complex translocates into the nucleus to initiate the transcription of interferon-stimulated genes that promote antiviral states. IRF-9 through this functional role impacts a range of cellular responses including proliferation apoptosis and immune regulation. It affects the temporal and spatial aspects of gene expression in response to interferons.
Pathways
IRF-9 plays a significant role in both the Jak-STAT and interferon signaling pathways. These pathways are essential for the activation of genes involved in immune defense. Through its involvement in these pathways IRF-9 interacts with proteins such as STAT1 and STAT2 creating a bridge between extracellular signals and gene activation. This involvement ensures that cells can respond effectively to viral infections enhancing innate and adaptive immune responses.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Target data
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Advanced Validation
- 0
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Primary Antibodies
AB271043
Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55]
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Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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