Western blot analysis of extracts from various cell lines using Cbl-b (D3C12) Rabbit mAb.
Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human Cbl-b (hCbl-b-Myc/DDK; +) and Myc/DDK-tagged full-length human c-Cbl (hc-Cbl-Myc/DDK; +), using Cbl-b (D3C12) Rabbit mAb (upper) or DYKDDDDK Tag Antibody #2368 (lower).
|REACTIVITY||H M R Mk|
|MW (kDa)||125, 130|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Cbl-b (D3C12) Rabbit mAb recognizes endogenous levels of total Cbl-b protein. This antibody does not cross-react with c-Cbl and based upon sequence alignment, is not predicted to cross-react with Cbl-c.Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human Cbl-b protein.
The Casitas B lineage lymphoma (Cbl) proteins (in mammals these are c-Cbl, Cbl-b, and Cbl-c) are a family of single subunit RING finger protein-ubiquitin E3 ligases that contain multiple protein interaction motifs (1). All Cbl proteins have a highly conserved N-terminal tyrosine kinase-binding (TKB) domain that mediates interactions between Cbl proteins and phosphorylated tyrosine residues on Cbl substrates. C-terminal to the RING finger, Cbl proteins have proline-rich domains that mediate interactions with SH3 domain-containing proteins. Phosphorylated tyrosine residues mediate interactions with SH2 domain-containing proteins such as the p85 subunit of PI3K. These protein-protein interaction motifs allow Cbl family proteins to function as adaptor proteins (2). This adaptor function contributes to the E3-dependent activities of Cbl proteins by targeting specific substrates for ubiquitination and degradation. The adaptor function also contributes to non-E3-dependent activities, such as the recruitment of proteins involved in receptor tyrosine kinase internalization, localization of Cbl proteins to specific subcellular compartments, and activation of discrete signaling pathways (1).
Cbl-b is an E3 ubiquitin ligase with a domain organization nearly identical to that of c-Cbl. The role of Cbl-b in hematopoietic cell physiology is well documented. Cbl-b expression is important for the downregulation of TCR expression during antigen recognition (2). Cbl-b also acts as a potent negative regulator of the CD28 signaling cascade to Vav and Rac1 through its ability to ubiquitinate the p85 regulatory subunit of PI3K (3,4). As a critical regulator of clonal anergy in T lymphocytes, Cbl-b mRNA and protein are upregulated in T cells following calcium mobilization and calcineurin activation (5). Cbl-b-deficient T cells are resistant to anergy induction (5). The molecular events governing this phenotype are thought to be linked to defects in the ubiquitination of PLCγ1 and PKCθ since the degradation of these signaling molecules, which occurs following restimulation of wild-type anergic T cells, fails to occur in Cbl-b-deficient T cells (5).
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