Bethyl Laboratories, Inc.

Antibodies to Subunits of the Cullin-E3 Ligases

April 8, 2010

Overview

Ubiquitin-directed degradation by the 26S proteasome is central to the control and regulation of a multitude of cellular processes. The covalent attachment of polyubiquitin chains to target proteins is coordinated by three classes of enzymes: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E1 is responsible for activating ubiquitin and passing it on to E2. E2 then assembles with E3 and transfers ubiquitin to a substrate protein. Multiple rounds of ubiquitin transfer occur to form polyubiquitin chains on the target protein and flag it for destruction. The E3 ligases play a critical role in this process because they determine the substrate specificity of the ubiquitination reaction. The two major types of E3 ligases are the cullin-RING-finger ligases (CRLs) and the anaphase-promoting complex/cyclosome (APC/C). The core complex of CRLs is composed of a cullin-homology domain-containing protein, a RING-H2 protein, and an adapter protein which facilitates interaction with substrate receptors. Seven cullin proteins have been identified in vertebrates: Cul1, Cul2, Cul3, Cul4a, Cul4b, Cul5, and Cul7. Studies indicate that each cullin protein associates with specific bridging adaptors and substrate receptors in the core complex to form substrate specific ligases. For example Cul1 associates with Skp1 and an F-box protein to form the SCF (Skp1, Cul1, F-box) ligase. In this complex, Skp1 bridges Cul1 to the F-box protein which functions as the substrate specific adaptor. Other Cul-based ligases include ECS (Elongin C, Cul2, SOCS), Cul4-DDB (DDB, Cul4, DCAF) and Cul3-BTB. A multitude of F-box, SOCS, BTB-domain, and DCAF factors have been identified and provide a critical means for assigning substrate specific activity to the E3 ligase function.

Selected Review
1. J. W. Harper, J. L. Burton, and M. J. Solomon, "The Anaphase-Promoting Complex: It's Not Just for Mitosis Any More," Genes Dev. 16, no. 17 (2002): 2179-2206.
2. J. Lee and P. Zhou, "DCAFs, the Missing Link of the CUL4-DDB1 Ubiquitin Ligase," Mol Cell. 26, no. 6 (2007): 775-780.
3. L. Pintard, A. Willems, and M. Peter, "Cullin-Based Ubiquitin Ligases: Cul3-BTB Complexes Join the Family," EMBO J. 23, no. 8 (2004): 1681-1687.

Bethyl Laboratories' Portfolio
AMBRA1 COP1/RFWD2 Cul3 Cul4a Cul5 Cul7 DDB1 DTL/CDT2 FBW7 FBXO28 FBXO31 FBXO38 LIS-1 NRIP PHIP RBBP4/RbAp48 RbBP5 RbBP7 Senataxin VprBP WDR21A WDR26 WDR28/GRWD1 WDR42A WDR5 ZBTB40 ZBTB7/FBI-1 ZNF295	Detection of Human and Mouse COP1/RFWD2 by WB (h&m) and IP (h). Samples: Whole cell lysate from HeLa (5, 15 and 50 mcg for WB; 1 mg for IP, 20% of IP loaded), 293T (T; 50 mcg), and mouse NIH3T3 (M; 50 mcg) cells. Antibodies: Affinity purified rabbit anti-COP1/RFWD2 antibody A300-894A used for WB at 0.04 mcg/ml (A) and 1 mcg/ml (B) and used for IP at 3 mcg/mg lysate (B). COP1/RFWD2 was also immunoprecipitated by rabbit anti-COP1/RFWD2 antibodies A300-895A and A300-896A, which recognize downstream epitopes. For blotting immunoprecipitated COP1/RFWD2 MAPK, ReliaBLOT^® Reagents and Procedures (Cat. No. WB120) were used. Detection: Chemiluminescence with exposure times of 3 minutes (A) and 10 seconds (B).	Detection of Human and Mouse DDB1 by WB (h&m);and by IP (h). Samples: Whole cell lysate from HeLa (5, 15, and 50 mcg for WB; 1 mg for IP), Ramos (R; 50 mcg) and mouse NIH3T3 (M; 50 mcg) cells. Antibodies: Affinity purified rabbit anti-DDB1 antibody BL1999 (Cat. No. A300-462A) used at 0.1 mcg/ml for WB (A and B) and at 3 mcg/mg lysate for IP. DDB1 was also immunoprecipitated using BL1996, which is an affinity purified antibody to an upstream epitope on DDB1. For blotting immunoprecipitated DDB1, ReliaBLOT^® Reagents and Procedures (Cat. No. WB120) were used. Detection: Chemiluminescence with exposure times of 10 seconds (A and B).

Search for Antibodies by Research Area
Bethyl Laboratories' portfolio of polyclonal antibodies commercially available includes over 5000 polyclonal antibodies to over 1500 proteins. We test our antibodies in a wide range of applications including ELISA, western blot, immunoprecipitation, immunohistochemistry, and immunocytochemistry. We have recently validated many of our antibodies for flow cytometry and ChIP. Our antibody range also includes secondary antibodies, loading controls and antibodies to epitope tags.

Bethyl Laboratories, Inc. - P.O. Box 850 - Montgomery, TX 77356
Phone: 1-800-338-9579 - Fax: 1-866-597-6105