CC0651

Kinase experiment:

For small-molecule screens, p27Kip1 is ubiquitinated in a 15 μL reaction with 40 nM phospho-p27, 40 nM E1, 5 μM E2, 25 nM SCFSkp2, 25 nM Cks1, and 27.8 μM biotinylated ubiquitin in 40 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 1 mM DTT, and 0.5 mM ATP at 23°C for 3 hr. The reaction is terminated with assay diluent, transferred to 384-well protein A plates coated with 62 ng SC528 antibody, incubated for 1 hr, and washed six times with 10 mM Tris-HCl [pH 7.6], 0.05% Tween20 prior to addition of 25 μL of 0.4 mg/mL europium streptavidin in HEPES [pH 7.6], 1% BSA, 0.2% Tween20 per well. Plates are incubated for 1 hr, washed, and read for Eu-time-resolved fluorescence after addition of 25 μL enhancement solution. For gel-based ubiquitination assays, CC0651 and its analogs are preincubated at the indicated concentrations with 0.5 μg E1, 1-4 μg hCdc34, and 50 ng SCFCdc4, 100 ng SCFFbw7, 150 ng SCFβTrCP, or 50 ng SCFSkp2 for 10-45 min at 4°C in 20 μL reaction buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2, 2 mM ATP, and 50μM DTT). Reactions are initiated by addition of 1μg ubiquitin and respective SCF substrates (50 ng HisSic1 phosphorylated by Cln2-Cdc28, 50ng cyclin E phosphorylated by Cdk2, 25 ng biotinylated IκBα phosphopeptide [KKERLLDDHDpSGLDpSMKDEE], or 50 ng p27Kip1 phosphorylated by cyclin E-Cdk2), incubated at 30°C for 1-3 hr, and products visualized by immunoblot[1].

Cell experiment:

PC-3 and HCT116 cell lines are grown in DMEM supplemented with Penicillin/Streptomycin, 2 mM glutamine, and 10% fetal bovine serum (FBS). Lentiviral shRNA constructs for human CDC34, UBE2R2, and nontarget control are used. For proliferation assays, PC-3 cultures are seeded at 5000 cells/well in quadruplicate in 24-well plates, grown for 24 hr, and then infected with lentiviral supernatant. For small-molecule inhibition, cells are seeded in quadruplicate at 500 cells/well in 96-well plates, grown for 24 hr, and treated with compound. Proliferation is measured by MTT assay. PC-3 or HCT 116 cultures are synchronized in G0/G1 by serum starvation for 30 hr and then released by addition of 10% serum. For epistasis experiments, PC-3 cells are infected with CDC34 shRNA for 24 hr, followed by addition of inhibitor and assessment of proliferation after 4 days. Anti-Cdc34, anti-p27, anti-α-tubulin, anti-ubiquitin, and anti-cyclin E antibodies are used[1].

References:

[1]. Ceccarelli DF, et al. An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme. Cell. 2011 Jun 24;145(7):1075-87.
[2]. Huang H, et al. E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin. Nat Chem Biol. 2014 Feb;10(2):156-63.