Suc llvy amc

The screening of compounds was performed at 1 μM final concentrations in the assay buffer (0.01% SDS, 50 mM Tris-HCl, 0.5 mM EDTA, pH 7.4). Stock solutions of compounds were prepared in DMSO. To 50 μL of each compound, 25 μL of 0.8 nM human iCP or human cCP (both from Boston Biochem, Inc., Cambridge, MA, USA) was added. After 30 min incubation at 37 °C, the reaction was initiated by the addition of 25 μL of 100 μM relevant fluorogenic substrate: acetyl-Nle-Pro-Nle-Asp-AMC (Ac-nLPnLD-AMC, [Bachem, Bubendorf, Switzerland]) for β1, acetyl-Pro-Ala-Leu-7-amino-4-methylcoumarin (Ac-PAL-AMC, [Boston Biochem, Inc., Cambridge, MA, USA]) for β1i, t-butyloxycarbonyl-Leu-Arg-Arg-7-amino-4-methylcoumarin (Boc-LRR-AMC, [Bachem, Bubendorf, Switzerland]) for β2 and β2i, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC [Bachem, Bubendorf, Switzerland]) for β5 and β5i. The reaction progress was recorded on the BioTek Synergy HT microplate reader by monitoring fluorescence at 460 nm (λ_ex = 360 nm) for 90 min at 37 °C. The initial linear ranges were used to calculate the velocity and to determine the residual activity.
In the case of the β1, β1i, β2, and β2i activity inhibition determination, the assay buffer was modified; SDS was replaced with the proteasomal activator PA28α (Boston Biochem, Inc., Cambridge, MA, USA).

The compounds selected by virtual ligand screening were purchased from ChemBridge corporation (www.chembridge.com). Purified human constitutive 20S proteasome cCP and human 20S immunoproteasome iCP from erythrocyte were obtained from Boston Biochem (Cambridge, USA). All fluorogenic substrates (Suc-LLVY-AMC, Boc-LRR-AMC, and Z-LLE-βNA) were obtained from Bachem (Weil am Rhein, Germany). Other reagents and solvents were purchased from commercial sources. Fluorescence was measured using a BMG Fluostar microplate reader (black 96-well microplates).

Carfilzomib and bortezomib were obtained from LC laboratories, ONX-0914 was obtained from MedChemExpress. LU-102, LU-015i, and activity-based probes were kindly provided by Drs. Bogdan Florea and Herman Overkleeft (Univ of Leiden, the Netherlands). Suc-LLVY-amc was obtained from Bachem. All other substrates were custom synthesized by China Peptide.

MDA-MB-231, HCC1143, and HCC1937 breast cancer cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Hep3B, Huh7, LCSC, HepG2, and PLC/PRF/5 hepatic cancer cells were a kind gift of Dr. Roberto Gedaly (College of Medicine, University of Kentucky). All other established cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). All cells were cultured according to the manufacturer’s protocol in 5% CO₂ in medium. Cultured cell lines were tested for Mycoplasma contamination routinely every 6 months. Specifically, H23 and H727 cells were tested twice in the course of performing the experiments described within this publication (Supplementary Fig. S4). Inhibitors of UPS pathways used in this study were purchased from commercial vendors: carfilzomib (LC Laboratories, Woburn, MA), bortezomib (ChemieTek, Indianapolis, IN), MG-132 (EMD Millipore, San Diego, CA), PYR-41 (ApexBio, Houston, TX), and P5091 (ApexBio, Houston, TX). The following proteasome fluorogenic substrates were used: Suc-LLVY-AMC (Bachem, Torrance, CA; I-1395), Ac-WLA-AMC (Boston Biochem, Cambridge, MA; S-330), Ac-nLPnLD-AMC (Bachem; I-1850), Ac-RLR-AMC (Boston Biochem; S-290), Ac-ANW-AMC (Boston Biochem; S-320), and Ac-PAL-AMC (Boston Biochem; S-310). Human recombinant Interferon-γ was purchased from eBioscience (San Diego, CA).