IU1(IC50=4.7 μ M), a reversible, specific human USP14 proteasome inhibitor, can penetrate the cell.

USP14:4.7 μM

High-throughput screening: Screening is conducted at the ICCB-Longwood screening facility. 10 μL of recombinant USP14 protein are dispensed into each well of a 384-well low volume plate in duplicate, using a Wellmate plate dispenser. 33.3?nL of compound from the library are pin-transferred into the wells using a Seiko pin transfer robotic system, followed by pre-incubation for about 30?min. The last two columns of each plate are used for positive and negative controls for the assay. To initiate the enzyme reaction, 10?μL of VS-proteasome plus Ub-AMC mixture are added to each well, using a Wellmate dispenser. Samples are then incubated for another 45?min. Ub-AMC hydrolysis is measured at Ex355/Em460 using an Envision plate reader. The final concentrations of USP14, VS-proteasome and Ub-AMC are 15?nM, 1?nM and 0.8 μM, respectively. The final concentration of test compound is approximately 17?μM. Enzymes and substrates are prepared in Ub-AMC assay buffer (50?mM Tris-HCl (pH?7.5), 1?mM EDTA, 1?mM ATP, 5 mM MgCl2, 1?mM DTT, and 1 mg/Ml ovalbumin).

MTT(Only for Reference)