Polyclonal and Monoclonal Antibodies Specific for Ubiquitin-specific Protease 20 Jang-Joon Park, Ji–Hyun Yun, and Kwang-Hyun Baek Ubiquitination and deubiquitination are important processes for numerous intracellular mechanisms, and the imbalance of these two processes can cause severe diseases including cancer. Accordingly, deubiquitinating enzymes (DUBs) responsible for deubiquitination from their protein substrates become attractive targets for many studies. USP20, also known as VDU2, belongs to ubiquitin-specific protease (USP) subfamily of DUBs and has several important roles in cells as shown with other DUBs. USP20 stabilizes HIF-1a by abolishing von Hippel-Lindau protein (pVHL)-E3 ligase complex-mediated HIF-1a degradation. USP20 is also associated with b 2 adrenergic receptor recycling. In addition, a previous study demonstrated that USP20 regulates Tax-induced NF-kB activation through its deubiquitinating activity. These studies provide a line of evidence that USP20 has critical roles in cellular functions. In this study, we generated and characterized a polyclonal and two mono- clonal antibodies against USP20. It is feasible that USP20 antibodies can be useful to investigate USP20-related cellular mechanisms and to find novel substrates of USP20. Introduction U biquitination has pivotal roles for many cellular processes, including cell cycle regulation, growth and differentiation, oncogenesis, pre-implantation, and tran- scriptional activation. This process is mediated by successive enzymatic actions of ubiquitin-activating enzymes (E1), ubi- quitin-conjugating enzymes (E2), and ubiquitin ligases (E3). In addition, E4 enzymes are needed for efficient ubiquitina- tion in some cases. (1) Ubiquitinated proteins are regulated depending on the different linkage formation of ubiquitin chains on one (or more) of seven lysine residues (K6, K11, K27, K29, K33, K48, and K63). K48-linked polyubiquitination is marked for 26S proteasomal degradation. On the other hand, K63-linked polyubiquitination has non-proteolytic functions including signaling transduction. (2,3) Formation of ubiquitin chains on other lysine residues is also responsible for various cellular functions. However, the detailed information on dif- ferent ubiquitin chain types is poorly understood. This sug- gests that the ubiquitination system is one of the dynamic regulators in cells. (4) Deubiquitination is a reversible process against ubiquiti- nation, which deconjugates ubiquitins from ubiquitinated proteins via enzymatic activity of deubiquitinating enzymes (DUBs). Until now, approximately 100 human genes encod- ing DUBs have been identified, and these DUBs can be grouped into at least five subfamily members: ubiquitin C- terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), otubain domain-containing proteins (OTUs), Josephin- containing proteins (MJDs), and JAB1/MPN/MOV34 me- talloenzymes ( JAMMs). (5,6) DUBs have conserved domains, including Cys, Asp/Asn, and His domains, which are asso- ciated with their catalytic activity. In addition, they have conserved motifs depending on each subfamily members. (7,8) These structural characteristics of DUBs are related to not only the capacity to hydrolyze the isopeptide bond of ubiquitin chains from ubiquitinated proteins, thereby preventing pro- teasomal degradation or altering cellular functions of ubi- quitinated proteins, but also the diverse protein-protein interactions and recognition of their substrates. (9,10) Thus, the coordination of ubiquitination and deubiquitination medi- ated by the actions of E3 ligases and DUBs is an important post-translational modification for homeostasis in cellular system, and malfunction of one of these processes can cause severe diseases including cancer. USP20 is known as VDU2 due to its property to interact with von Huppel-Lindau protein (pVHL) as well as USP33 (VDU1). (11) It interacts with and stabilizes HIF-1a through its deubiquitinating activity, thereby counteracting the pVHL- mediated ubiquitination process and regulating HIF-1a pro- tein levels. In addition, USP20 and USP33 have the ability to deubiquitinate b 2 adrenergic receptor (b 2 AR) and control b 2 AR functions for lysosomal membrane trafficking. (12) Ber- thouze and colleagues showed that b 2 AR activation by its agonist induces ubiquitination along with lysosomal degra- dation of receptor, and USP20 and USP33 overexpression causes deubiquitination of ubiquitinated receptor and en- hances receptor recycling and resensitization. (12) Further Department of Biomedical Science, CHA University, CHA General Hospital, Seongnam, Republic of Korea. MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 32, Number 3, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2012.0120 193