Human LRP6 knockout Hep G2 cell line (ab277909)
Overview
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Product name
Human LRP6 knockout Hep G2 cell line -
Parental Cell Line
HepG2 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 46 bp deletion in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type Hep G2 cell line (ab275467). 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: MEM + 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
Liver -
Cell type
epithelial -
Disease
Hepatocellular Carcinoma -
Gender
Male -
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
Component of the Wnt-Fzd-LRP5-LRP6 complex that triggers beta-catenin signaling through inducing aggregation of receptor-ligand complexes into ribosome-sized signalsomes. Cell-surface coreceptor of Wnt/beta-catenin signaling, which plays a pivotal role in bone formation. The Wnt-induced Fzd/LRP6 coreceptor complex recruits DVL1 polymers to the plasma membrane which, in turn, recruits the AXIN1/GSK3B-complex to the cell surface promoting the formation of signalsomes and inhibiting AXIN1/GSK3-mediated phosphorylation and destruction of beta-catenin. Required for posterior patterning of the epiblast during gastrulation. -
Tissue specificity
Widely co-expressed with LRP5 during embryogenesis and in adult tissues. -
Involvement in disease
Defects in LRP6 are the cause of autosomal dominant coronary artery disease type 2 (ADCAD2) [MIM:610947]. -
Sequence similarities
Belongs to the LDLR family.
Contains 4 EGF-like domains.
Contains 3 LDL-receptor class A domains.
Contains 20 LDL-receptor class B repeats. -
Domain
The YWTD-EGF-like domains 1 and 2 are required for the interaction with Wnt-frizzled complex. The YWTD-EGF-like domains 3 and 4 are required for the interaction with DKK1.
The PPPSP motifs play a central role in signal transduction by being phosphorylated, leading to activate the Wnt signaling pathway. -
Post-translational
modificationsDual phosphorylation of cytoplasmic PPPSP motifs sequentially by GSK3 and CK1 is required for AXIN1-binding, and subsequent stabilization and activation of beta-catenin via preventing GSK3-mediated phosphorylation of beta-catenin. Phosphorylated, in vitro, by GRK5/6 within and outside the PPPSP motifs. Phosphorylation at Ser-1490 by CDK14 during G2/M phase leads to regulation of the Wnt signaling pathway during the cell cycle. Phosphorylation by GSK3B is induced by RPSO1 binding and inhibited by DKK1. Phosphorylated, in vitro, by casein kinase I on Thr-1479.
Undergoes gamma-secretase-dependent regulated intramembrane proteolysis (RIP). The extracellular domain is first released by shedding, and then, through the action of gamma-secretase, the intracellular domain (ICD) is released into the cytoplasm where it is free to bind to GSK3B and to activate canonical Wnt signaling.
Palmitoylation on the two sites near the transmembrane domain leads to release of LRP6 from the endoplasmic reticulum.
Mono-ubiquitinated which retains LRP6 in the endoplasmic reticulum.
N-glycosylation is required for cell surface location. -
Cellular localization
Membrane. Endoplasmic reticulum. On Wnt signaling, undergoes a cycle of caveolin- or clathrin-mediated endocytosis and plasma membrane location. Released from the endoplasmic reticulum on palmitoylation. Mono-ubiquitination retains it in the endoplasmic reticulum in the absence of palmitoylation. On Wnt signaling, phosphorylated, aggregates and colocalizes with AXIN1 and GSK3B at the plasma membrane in LRP6-signalsomes. Chaperoned to the plasma membrane by MESD. - Information by UniProt
Associated products
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab277909 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 |
---|---|---|
WB |
Use at an assay dependent concentration. Predicted molecular weight: 180 kDa.
|
Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 180 kDa. |
Images
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All lanes : Anti-LRP6 antibody [EPR22910-39] (ab231779) at 1/1000 dilution
Lane 1 : Wild-type HepG2 cell lysate
Lane 2 : LRP6 knockout HepG2 cell lysate
Lane 3 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 180 kDa
Observed band size: 180,200 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-LRP6 antibody [EPR22910-39] 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, ab231779 was shown to bind specifically to LRP6. A band was observed at 180/200 kDa in wild-type HepG2 cell lysates with no signal observed at this size in LRP6 knockout cell line ab277909 (knockout cell lysate ab284222). To generate this image, wild-type and LRP6 knockout HepG2 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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All lanes : Anti-LRP6 antibody [EPR22910-39] (ab231779) at 1/1000 dilution
Lane 1 : Wild-type HepG2 cell lysate
Lane 2 : LRP6 knockout HepG2 cell lysate
Lane 3 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 180 kDa
Observed band size: 180,200 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-LRP6 antibody [EPR22910-39] 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, ab231779 was shown to bind specifically to LRP6. A band was observed at 180/200 kDa in wild-type HepG2 cell lysates with no signal observed at this size in LRP6 knockout cell line ab277909 (knockout cell lysate ab284222). To generate this image, wild-type and LRP6 knockout HepG2 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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46 bp deletion in exon 2
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab277909 has not yet been referenced specifically in any publications.