Optiplate

The recombinant DUBs were captured with anti-AGIA tag-conjugated magnetic beads, using the same procedure as in the in vitro DUB assay. The recombinant DUB on the magnetic beads was combined with 10 µL of substrate mixture containing 2 µM of substrate ubiquitins shown in Figure S5, in 50 mM Tris-HCl, pH 7.5, 5 mM DTT, and the deubiquitination assay was performed for 3 h at 30 °C. The supernatant was separated from the recombinant DUB on the beads by a magnetic stand, and a 5 µL portion of each reaction was transferred to a 384-well OptiPlate (PerkinElmer, Waltham, MA, USA) containing AlphaScreen beads mix (100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mg/mL BSA, 0.1% Tween 20), 7.5 ng anti-DYKDDDDK (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 0.08 µL streptavidin-conjugated donor beads, and 0.08 µL protein A-coated acceptor beads (PerkinElmer), in a total volume of 25 µL. The OptiPlate was incubated for 1 h at room temperature, and the luminescent signal was detected by an EnVision plate reader (PerkinElmer).

The binding interactions between biotinylated p53 and FLAG-tagged E3s were detected with AlphaScreen technology provided by PerkinElmer. Fourteen microliters of biotinylated protein mixture containing 100 mM Tris-HCl, pH 7.5, 1 mg/mL bovine serum albumin, 0.1% Tween 20, 100 mM NaCl, and 0.75 μL crude biotinylated protein was dispensed into a 384-well OptiPlate (PerkinElmer, Waltham, MA, USA). Then, 0.75 μL crude FLAG-protein was added to the individual wells of the OptiPlate containing the biotinylated protein mixture and the plate was incubated at 26°C. After one hour incubation, a detection mixture containing 100 mM Tris-HCl, pH 7.5, 1 mg/mL bovine serum albumin, 0.1% Tween 20, 100 mM NaCl, 0.2 μg/mL Anti-DYKDDDDK antibody (1E6, Wako), 0.1 μL streptavidin-coated donor beads, and 0.1 μL protein A-conjugated acceptor beads was added to each well of the OptiPlate and the plate was incubated at 23°C for 1 h. Luminescence was analyzed with the AlphaScreen detection program. For the screening with the E3 protein array, biotinylated bait proteins were dispensed to OptiPlate by a FlexDrop dispenser (PerkinElmer) and then each FLAG-tagged E3 in 96-well titer plate was mixed with the biotinylated protein in the OptiPlate using Janus dispenser (PerkinElmer).

For the initial screen, we used a total of 412 complementary DNA library encoding human protein kinases^{35 (link),36 (link)}. DNA templates containing a biotin-ligating sequence were amplified by split-PCR with cDNAs and corresponding primers, and then used with the GenDecoder protein production system (CellFree Science)^{37 (link)–39 (link)}. For synthesis of Vpx protein, vpx genes derived from the pGL-AN proviral plasmid^{40 (link)} were generated by split-PCR and used as DNA templates. FLAG-tagged Vpx proteins were mixed with biotinylated kinases in 15 µl of reaction buffer (20 mM Tris–HCl pH 7.6, 5 mM MgCl₂, 1 mM DTT) in 384-well OptiPlates (PerkinElmer) and incubated at 26 °C for 1 h. Each sample was then mixed with AlphaScreen buffer containing anti-immunoglobulin G (protein A) acceptor beads and streptavidin-coated donor beads (0.1 µl each; PerkinElmer) and the anti-FLAG M2 antibody (5 µg ml⁻¹; Sigma-Aldrich), and further incubated at 26 °C. One hour later, AlphaScreen signals from the mixture were detected on an EnVision device (PerkinElmer) using the AlphaScreen signal detection program.