Human STAT3 knockout HeLa cell line
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human STAT3 knockout HeLa cell line (AB255436)
Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.
Lanes 1-4 : Merged signal (red and green). Green - ab267373 observed at 88 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.
Lanes 1-2 : ab267373 Anti-STAT3 (phospho Y705) antibody [EPR23968-52] was shown to specifically react with STAT3 in wild-type serum starved and then IFN alpha treated HeLa cells. Loss of signal was observed when serum starved and then IFN alpha treated knockout cell line ab255436 (knockout cell lysate ab263797) was used. Wild-type and STAT3 knockout samples were subjected to SDS-PAGE. ab267373 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4° overnight 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 10000 dilution for 1 hour at room temperature before imaging.
Lysates loaded onto lanes 3-4 were made freshly and used in WB immediately to minimize protein degradation.
All lanes:
Western blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] (<a href='/en-us/products/primary-antibodies/stat3-phospho-y705-antibody-epr23968-52-ab267373'>ab267373</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa (human cervix adenocarcinoma epithelial cell) serum starved overnight, then treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate at 20 µg
Lane 2:
STAT3 knockout HeLa serum starved overnight, then treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate at 20 µg
Lane 2:
Western blot - Human STAT3 knockout HeLa cell line (ab255436)
Lane 3:
Jurkat (human t cell leukemia cell line from peripheral blood) treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate at 20 µg
Lane 4:
HepG2 (human hepatocellar carcinoma epithelial cell) serum starved overnight, then treated with 100 ng/ml IL-6 for 30 minutes, whole cell lysate at 20 µg
Secondary
All lanes:
Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution
Predicted band size: 88 kDa
Observed band size: 88 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human STAT3 knockout HeLa cell line (AB255436)
Homozygous : 1 bp deletion in exon 2.
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AB68153
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![Western blot - Anti-STAT3 antibody [EPR787Y] (AB68153)](https://content.abcam.com/products/images/stat3-antibody-epr787y-ab68153--western-blot-img16701.jpg)
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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 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
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
STAT3 is an important player in regulating cell growth and apoptosis. It functions not only as a transcription factor but also as part of a larger protein complex that operates within the cell nucleus to influence gene expression. This multifaceted role enables STAT3 to control the expression of genes related to cell proliferation survival and differentiation impacting various biological processes. These functions highlight its importance in tissue homeostasis and response to extracellular signals.
Pathways
STAT3 is actively involved in the JAK-STAT signaling pathway a principal route for many cytokines and growth factors and the MAPK pathway. STAT3 interacts with proteins such as JAK kinases which phosphorylate Stat3 and initiate the STAT3 signaling cascade. Additionally it closely associates with the protein Raf1 in the MAPK pathway linking external signals to transcriptional responses. These pathways play a significant role in immune response inflammation and growth signaling.
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
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