Human WWTR1 knockout HeLa cell line (ab281598)
製品の概要
-
製品名
Human WWTR1 knockout HeLa cell line
TAZ ライゼート 製品一覧 -
Parental Cell Line
HeLa -
Organism
Human -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Biosafety level
2 -
特記事項
Recommended control: Human wild-type HeLa cell line (ab275466). 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.
製品の特性
-
Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
Mycoplasma free
Yes -
保存方法
Shipped on Dry Ice. Store in liquid nitrogen. -
バッファー
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
研究分野
ターゲット情報
-
機能
Transcriptional coactivator which acts as a downstream regulatory target in the Hippo signaling pathway that plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. The core of this pathway is composed of a kinase cascade wherein MST1/MST2, in complex with its regulatory protein SAV1, phosphorylates and activates LATS1/2 in complex with its regulatory protein MOB1, which in turn phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ. WWTR1 enhances PAX8 and NKX2-1/TTF1-dependent gene activation. Regulates the nuclear accumulation of SMADS and has a key role in coupling them to the transcriptional machinery such as the mediator complex. Regulates embryonic stem-cell self-renewal, promotes cell proliferation and epithelial-mesenchymal transition. -
組織特異性
Highly expressed in kidney, heart, placenta and lung. Expressed in the thyroid tissue. -
配列類似性
Contains 1 WW domain. -
ドメイン
The PDZ-binding motif is essential for stimulated gene transcription. It localizes the protein into both punctate nuclear foci and plasma membrane-associated complexes.
Binds to transcription factors via its WW domain. -
翻訳後修飾
Phosphorylated by LATS2 and STK3/MST2. Phosphorylation by LATS2 results in creation of 14-3-3 binding sites, retention in the cytoplasm, and functional inactivation. Phosphorylation results in the inhibition of transcriptional coactivation through YWHAZ-mediated nuclear export. -
細胞内局在
Nucleus. Cytoplasm. Concentrates along specific portions of the plasma membrane, and accumulates in punctate nuclear bodies. When phosphorylated, is retained in cytoplasm by YWHAZ. Can be retained in the nucleus by MED15. - Information by UniProt
画像
-
Sanger Sequencing - Human WWTR1 knockout HeLa cell line (ab281598)
-
Western blot - Human WWTR1 knockout HeLa cell line (ab281598)All lanes : Anti-TAZ antibody [CL0371] (ab242313) at 1 µg/ml
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : WWTR1 knockout HeLa cell lysate
Lane 3 : A549 cell lysate
Lane 4 : K562 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 52 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-TAZ antibody [CL0371] staining at 1 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab242313 was shown to bind specifically to TAZ. A band was observed at 52 kDa in wild-type HeLa cell lysates with no signal observed at this size in WWTR1 knockout cell line ab281598 (knockout cell lysate ab282950). To generate this image, wild-type and WWTR1 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
-
Western blot - Human WWTR1 knockout HeLa cell line (ab281598)All lanes : Anti-TAZ antibody (ab110239) at 1 µg/ml
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : WWTR1 knockout HeLa cell lysate
Lane 3 : A549 cell lysate
Lane 4 : K562 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 52 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-TAZ antibody staining at 1 ug/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab110239 was shown to bind specifically to TAZ. A band was observed at 52 kDa in wild-type HeLa cell lysates with no signal observed at this size in WWTR1 knockout cell line ab281598 (knockout cell lysate ab282950). To generate this image, wild-type and WWTR1 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
プロトコール
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
データシートおよび資料
-
SDS download
-
Datasheet download
参考文献 (0)
ab281598 は論文での使用が確認できていません。