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AB267245

Human CBL knockout HEK-293T cell line

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CBL KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: 10 bp deletion in exon 9. To order both knockout and wild-type control cells: select '2 x 1000000 Cells/vial'. To order only knockout cells: select '1000000 Cells/vial'.
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Western blot - Human CBL knockout HEK-293T cell line (AB267245)
  • WB

Lab

Western blot - Human CBL knockout HEK-293T cell line (AB267245)

Lanes 1-4 : Merged signal (red and green). Green - ab32027 observed at 110 kDa. Red - loading control ab8245 observed at 36 kDa.

Unpurified ab32027 Anti-CBL antibody [YE323] - C-terminal was shown to specifically react with CBL in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267245 (knockout cell lysate ab257200) was used. Wild-type and CBL knockout samples were subjected to SDS-PAGE. ab32027 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° 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 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CBL antibody [YE323] - C-terminal (<a href='/en-us/products/primary-antibodies/cbl-antibody-ye323-c-terminal-ab32027'>ab32027</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

CBL knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human CBL knockout HEK-293T cell line (ab267245)

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

Raji cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 100 kDa

Observed band size: 110 kDa

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Sanger Sequencing - Human CBL knockout HEK-293T cell line (AB267245)
  • Sanger seq

Unknown

Sanger Sequencing - Human CBL knockout HEK-293T cell line (AB267245)

Homozygous : 10 bp deletion in exon9

Cell Culture - Human CBL knockout HEK-293T cell line (AB267245)
  • Cell Culture

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Cell Culture - Human CBL knockout HEK-293T cell line (AB267245)

Representative images of CBL knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.

Immunocytochemistry/ Immunofluorescence - Human CBL knockout HEK-293T cell line (AB267245)
  • ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Human CBL knockout HEK-293T cell line (AB267245)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CBL KO HEK293T (CBL knockout human embryonic kidney epithelial cell), ab267245 cells labelling CBL with ab324458 at 1/100 (4.71 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in wildtype HEK-293T, and showing no staining in CBL knockout HEK-293T cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

Western blot - Human CBL knockout HEK-293T cell line (AB267245)
  • WB

Supplier Data

Western blot - Human CBL knockout HEK-293T cell line (AB267245)

Blocking and diluting buffer and concentration : 5% NFDM/TBSTLow expression : NIH/3T3 (PMID : 12193575). Performed under reducing conditions. Frozen lysates were used for Western blotting. In Western blot, ab309549 was shown to bind specifically to CBL. Target of interest was observed at 120 kDa wild-type 293T cell lysates (lane 1) with no signal observed at this size in CBL knockout cell line (lane 2, knockout cell line ab267245 / knockout cell lysate ab257200). The band beneath the target band (120 kDa) is likely to be degraded target fragment. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 3 minutes

All lanes:

Western blot - Anti-CBL antibody [17/c-Cbl] (<a href='/en-us/products/primary-antibodies/cbl-antibody-17-c-cbl-ab309549'>ab309549</a>) at 1/1000 dilution

Lane 1:

Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CBL knockout HEK-293T cell line (ab267245) at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) (ZB-2305) at 1/10000 dilution

Observed band size: 120 kDa

false

Exposure time: 3min

Key facts

Cell type

HEK-293T

Species or organism

Human

Tissue

Kidney

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: 10 bp deletion in exon 9

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Complementary Products

Primary Antibodies

AB32027

Anti-CBL antibody [YE323] - C-terminal

20ul selling size|RabMAb|Recombinant|KO Validated

Flow Cytometry (Intracellular) - Anti-CBL antibody [YE323] - C-terminal (AB32027)

  • 0
  • 0
Primary Antibodies

AB92312

Anti-MLH1 antibody [EPR3894]

RabMAb|Recombinant|KO Validated|20ul selling size

Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR3894] (AB92312)

  • 5
  • 4
Cell Lines & Lysates

AB267324

Human ATG4C knockout HEK-293T cell line

Advanced Validation

Western blot - Human ATG4C knockout HEK-293T cell line (AB267324)

  • 0
  • 0
Primary Antibodies

AB92471

Anti-MSH6 antibody [EPR3945]

BOND RX™ Validated|RabMAb|Recombinant|KO Validated|Trial Size

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MSH6 antibody [EPR3945] (AB92471)

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  • 5
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Reactivity data

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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.

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What's included?

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Properties and storage information

Gene name
CBL
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Zygosity
Homozygous
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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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.

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

The CBL protein also known as C-cbl or Casitas B-lineage lymphoma proto-oncogene serves as an E3 ubiquitin-protein ligase. This protein with a molecular mass of approximately 120-130 kDa plays a critical role in the regulation of receptor tyrosine kinases. It facilitates the polyubiquitination of activated receptor proteins targeting them for degradation in the proteasome. CBL is predominantly expressed in various tissues including the hematopoietic system. It is a significant component in the control of cell signaling and helps maintain cellular homeostasis.
Biological function summary

The CBL protein acts as a negative regulator for several signaling pathways. It integrates into signaling complexes and modulates the activation of pathways by interacting with other proteins. CBL does not act alone; it commonly forms complexes with proteins such as Grb2 and p85 influencing downstream signaling influence in pathways. This regulation prevents excessive activation of signals that can lead to cellular proliferation or differentiation abnormally.

Pathways

CBL integrates into the ubiquitin-proteasome system and the Ras signaling pathway. Within these pathways CBL works to modulate cell surface receptors ensuring that signaling threshold is not surpassed. The importance of CBL in the Ras signaling pathway is highlighted by its interactions with proteins like Ras-GAP and SHP2. Through these interactions CBL controls the rate of signal transduction affecting cellular responses such as growth survival and motility.

CBL involvement has been linked to several forms of cancer and immune deficiencies. Mutations or dysregulation of the CBL gene can lead to myelodysplastic syndromes and acute myeloid leukemia. In these conditions the interaction between CBL and other proteins such as JAK2 becomes dysregulated driving abnormal cell growth and survival. Furthermore CBL's failure to properly degrade tyrosine kinase receptors can contribute to enhanced signaling fostering oncogenic processes.
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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 2 US: 2

Adherent/suspension

Adherent

Gender

Female

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Product protocols

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Product promise

We are committed to supporting your work with high-quality reagents, and we're here for you every step of the way. In the unlikely event that one of our products does not perform as expected, you're protected by our Product Promise.
For full details, please see our Terms & Conditions

Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.

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