Cbl (named after Casitas B-lineage Lymphoma) is a mammalian gene family. CBL gene, a part of the Cbl family, encodes the protein CBL which is an E3 ubiquitin-protein ligase involved in cell signalling and protein ubiquitination. Mutations to this gene have been implicated in a number of human cancers, particularly acute myeloid leukaemia. Ubiquitination is the process of chemically attaching ubiquitin monomers to a protein, thereby targeting it for degradation. As this is a multi-step process, several different enzymes are involved, the final one being a member of the E3 family of ligases. Cbl functions as an E3 ligase, and therefore is able to catalyse the formation of a covalent bond between ubiquitin and Cbl's protein substrate - typically a receptor tyrosine kinase. The RING-finger domain mediates this transfer, however like other E3 ligases of the RING type no intermediate covalent bond is formed between ubiquitin and the RING-finger domain. The stepwise attachment of ubiquitin to the substrate receptor tyrosine kinase can lead to its removal from the plasma membrane and subsequent trafficking to the lysosome for degradation.
Background References
1. Leardini D et al. Role of CBL Mutations in Cancer and Non-Malignant Phenotype. Cancers (Basel). 2022 Feb
2. Pinilla-Macua I et al. Cbl and Cbl-b independently regulate EGFR through distinct receptor interaction modes. Mol Biol Cell. 2023 Dec
Western blot analysis of CBL on different lysates with Rabbit anti-CBL antibody (HA723936) at 1/5,000 dilution.
Lane 1: Raji cell lysate (20 µg/Lane) Lane 2: K-562 cell lysate (20 µg/Lane) Lane 3: COS-1 cell lysate (20 µg/Lane) Lane 4: Mouse testis tissue lysate (30 µg/Lane) Lane 5: Rat testis tissue lysate (30 µg/Lane)
Predicted band size: 100 kDa Observed band size: 110 kDa
Exposure time: 20 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723936) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunocytochemistry analysis of Raji cells labeling CBL with Rabbit anti-CBL antibody (HA723936) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CBL antibody (HA723936) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Flow cytometric analysis of Raji cells labeling CBL.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA723936, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
CBL was immunoprecipitated from 0.2 mg Raji cell lysate with HA723936 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723936 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: Raji cell lysate (input) Lane 2: HA723936 IP in Raji cell lysate Lane 3: Rabbit IgG instead of HA723936 in Raji cell lysate