ain of GrsA with an N-terminal 66His tag to 14192894 generate the plasmid pGEX-PCP-APPBP1-Uba3. This plasmid was used for the coexpression of the PCP-APPBP1 fusion and Uba3 to assemble PCP-NAE. For the expression of HA-Nedd8 with an N-terminal HA tag, the Nedd8 gene was amplified by polymerase chain reaction from pGEX-NEDD8 and cloned into pET28a between the NheI and NotI restriction sites. The pET-HA-Nedd8 plasmid expressed HA-Nedd8 fusion with an N-terminal 66His tag. Protein Expression and Purification For the expression of proteins with a 66His tag from the pET or pGEX vectors, the plasmids were transformed into the BL21pLysS chemical competent cells and plated on LB-agar plates with appropriate antibiotics. Protein expression and purification followed the protocol provided by the vendors of the pET expression system and the Ni-NTA agarose resin. Typically the cells were grown in 1 L Lysogeny Broth supplemented with 100 mg/mL ampicillin to an OD around 0.60.8 at 37uC. The LB culture was then induced by adding IPTG to a final concentration of 1 mM and incubating with continuous shaking at 15uC overnight. Cells were subsequently collected by centrifugation at 5,000 rpm for 10 min, resuspended in lysis buffer and lysed with French press. The resulting crude suspension was centrifuged at 12,000 rpm to remove the pelleted cell debris from protein lysate. The lysate supernatant was mixed with 1 mL of Ni-NTA agarose and rocked gently at 4uC for 2 hours. 
The slurry was next transferred to a gravity column, washed once with 15 mL lysis buffer, twice with 15 mL wash buffer and eluted with 5 mL elution buffer. Eluted protein was dialyzed overnight at 4uC in a 1 L buffer, followed by a second dialysis the next day with the same buffer for 3 hours. All purified proteins were assayed by electrophoresis on a 4 15% SDS Tris polyacrylamide gel for the verification of their sizes and purity, and eventually stored in aliquots at 280uC. Ubc12 and the cullin3-Rbx1 complex was expressed and purified as previously reported. Biotin Conjugation to PCP-NAE Fusion Biotin labeling of PCP-NAE catalyzed by Sfp phosphopantetheinyl transferase followed a reported protocol. 100 mL labeling reaction was set up containing 5 mM PCP-NAE, 2 mM biotin-CoA, 0.3 mM Sfp in a reaction buffer. The reaction was allowed to proceed for 1 hour at 30uC, and then mixed with 100 mL 3% BSA. 100 mL of the reaction mixture was distributed to a 96-well plate coated with Varlitinib biological activity streptavidin and allowed to bind to the plate for 1 hour at room temperature. The plate was then washed three times with TBS buffer to remove unbound enzymes before the phage selection reaction. Materials and Methods Molecular Cloning Enzyme-linked Immunosorbent Assay The transfer of Nedd8 to biotin labeled PCP-NAE bound to the streptavidin plate was analyzed by ELISA. After the labeling reaction, 22634634 PCP-NAE attached with biotin was bound to a streptavidin plate and the plate was washed with TBS. 5 mM Nedd8 protein with an N-terminal HA tag was added to the Nedd8-Like Ubiquitin Variants streptavidin plate in the presence of 5 mM ATP in TBS. Control reactions were also set up excluding ATP or using a streptavidin plate without the coating of PCP-NAE. After a 1 hour incubation, the plate was washed and the Nedd8 protein bound to the plate was detected by binding consecutively with a mouse anti-HA antibody and a goat anti-mouse antibody conjugated with HRP . The peroxidase activity immobilized on the plate was assayed by the addition o
