Cullin-associated and neddylation-dissociated (CAND1)/TIP120A is a protein containing multiple HEAT repeats. It functions, in part, as an inhibitor of multiple cullin-RING ubiquitin ligases (CRLs) via binding to cullin-RBX complexes that are both unconjugated to NEDD8 and lack association with substrate recognition subunits (1-3). Indeed, CAND1 has been shown to bind all cullin family members in human cells and analysis of the crystal structure of human CAND1 bound to the CUL1-RBX1 complex suggests that CAND1 inhibits the activity of CRLs by sterically blocking both the substrate recognition subunit binding site and the NEDD8 conjugation site . Conversely, CAND1 binding to cullin-RBX complexes is incompatible with neddylation as NEDD8 conjugated to cullins blocks CAND1 binding, suggesting that CAND1 binds to cullins only after the COP9 signalosome has catalyzed cullin deneddylation. Through its ability to negatively regulate CRL assembly, CAND1 plays an integral part in facilitating CRL activation cycles that allow CRLs to utilize distinct substrate recognition subunits and protects these subunits from undergoing ubiquitin-dependent degradation .
Background References
1. Politis PK et al. BM88/CEND1 coordinates cell cycle exit and differentiation of neuronal precursors. Proc Natl Acad Sci U S A 104(45):17861-17866 (2007).
Cell cycle exit and neuronal differentiation 1 antibody
Cell cycle exit and neuronal differentiation protein 1 antibody
FLJ90066 antibody
MGC34326 antibody
Images
Western blot analysis of CEND1 on different lysates with Rabbit anti-CEND1 antibody (ET7109-75) at 1/5,000 dilution.
Lane 1: Rat brain tissue lysate Lane 2: Mouse brain tissue lysate Lane 3: Mouse cerebellum tissue lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 15 kDa Observed band size: 25 kDa
Exposure time: 2 minutes;
15% 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 (ET7109-75) at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
ICC staining of CEND1 in N2A cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET7109-75, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).
Immunohistochemical analysis of paraffin-embedded rat brain tissue using anti-CEND1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7109-75, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-CEND1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7109-75, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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