Ha ubvs

Bilirubin and biliverdin were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Other agents used include NEM (Sigma-Aldrich Inc.), the Proteasome-Glo Chymotrypsin-like Cell-Based Assay kit (Promega Bioscience, Madison, WI, USA), Suc-Leu-Leu-Val-Tyr-aminomethylcoumarin (Suc-LLVY-AMC), Boc-Leu-Arg-Arg- aminomethylcoumarin (Boc-LRR-AMC), Z-Leu-Leu-Glu-AMC, 20S and 26S human proteasome preparations, HA-Ub-VS, K48-linked tetraubiquitin, Ubiquitin-AMC (U550) (Boston Biochem, Cambridge, MA, USA), JC-1 (Beyotime, Shanghai, China) and MTS assay kit (CellTiter 96 Aqueous One Solution reagent) (Promega Corporation, Madison, WI, USA). Antibodies used in this study and their sources are: anti-ubiquitin (P4D1) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-K48-linkage specific polyubiquitin (D9D5), anti-Bax (D3R2M) (Cell Signaling Technology, Beverly, MA, USA), anti-GAPDH, anti-HA-tag (Bioworld Technology, Inc., Louis Park, MN, USA) and anti-Tau-1, (Invitrogen, Carlsbad, CA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from Santa Cruz Biotechnology Inc.

For assessment of DUB activity toward HA‐ubiquitin vinyl sulfone (HA‐Ub‐VS), cells were lysed in DUB activity buffer (50 mM Tris/HCl pH 7.4, 5 mM MgCl₂, 250 mM sucrose, 1 mM DTT and 2 mM ATP) for 20 min on ice and lysates subsequently clarified. Equal amounts of protein were mixed with 5 μM HA‐Ub‐VS (Boston Biochem #U‐212) and subsequently incubated at 37°C for 45 min. The reaction was stopped and lysates were denatured by the addition of 1% SDS and subsequent boiling at 95°C for 5 min. After cooling to room temperature, samples were diluted with DUB activity buffer to a final volume of 500 μl. Active DUBs modified by HA‐Ub‐VS were immunoprecipitated by HA agarose. Beads were washed four times with washing buffer (250 mM NaCl, 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 0.1% Triton X‐100, 50 mM NaF).
For in vitro DUB assays, FLAG‐tagged LIN28B was co‐expressed with HA‐tagged ubiquitin in HEK293T cells, purified as described for in vivo ubiquitylation experiments, eluted with 3xFLAG peptide (Sigma #F4799) at a final concentration of 1 mg/ml in DUB buffer (50 mM Tris/HCl pH 7.4, 100 mM NaCl, 2 mM DTT) and used as substrate. GST or GST‐OTUD6B bound to beads were washed three times with DUB buffer, activated for 10 min at 23°C and subsequently incubated with eluted substrate for 1.5 h at 37°C. The reaction was stopped by the addition of laemmli buffer and boiling at 95°C for 5 min.

BA and WP were obtained from Biovision (Milpitas, CA); QVD from R & D Systems (Minneapolis, MN); CsA and SP600125 from Enzo Life Sciences (Farmingdale, NY); Chx from Sigma-Aldrich (St. Louis, MO); Btz and U0126 from LC Laboratories (Woburn, MA); LY294002 from EMD Millipore (Billerica, MA); HA-UbVS from Boston Biochem (Cambridge, MA); Enz from Selleckchem (Houston, TX); Coomassie blue from EMD Biosciences (Temecula, CA); and trypan blue (0.4%) from Invitrogen (Carlsbad, CA). All other reagents were purchased from Sigma-Aldrich.

EOAI3402143 (referred to as G9) was synthesized and provided by Cheminpharma (Branford, CT). Other reagents used in this study were obtained from the following sources: hemagglutinin-tagged ubiquitin vinyl methyl sulfone (HA-UbVS; Boston Biochem); vemurafenib (PLX4032; Chemie Tek); PD 0325901 (Cayman Chemical). All reagents were made up and stored frozen as 10 mM stock solutions.

WM cells were treated with dimethyl sulfoxide (DMSO) or VLX1570 (250 nm) for 3 h, harvested and lysed in RIPA lysis buffer followed by centrifugation at 13 000  r.p.m. for 5 min. Total protein (20 μg) was labeled with 5 μm HA-tagged ubiquitin-vinyl sulfone (HA-Ub-VS; Boston Biochem, Cambridge, MA, USA) probe for 1, 10, 20 or 30 min at 37 °C and then subjected to western blotting with anti-USP14 or anti-UCHL5 antibodies.