K48 linked tetraubiquitin

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.

ZnPT, cisplatin and b-AP15 were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA); human 20S and 26S proteasomes, Suc-Leu-Leu-Val-Tyr-aminomethylcoumarin (Suc-LLVY-AMC), HA-Ubiquitin-Vinyl Sulfone (HA-Ub-VS), K48-linked tetra-ubiquitin and ubiquitin-AMC were purchased from Boston Biochem (Cambridge, MA, USA); bortezomib was purchased from BD Biosciences (San Jose, CA, USA). The sources of antibodies used in this study: anti-ubiquitin (P4D1), anti-p27 (F-8), anti-GFP (B-2) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-caspase3 (8G10), anti-caspase8 (1C12), anti-caspase9 (C9), anti-PARP, anti-p21 Waf1/Cip1 (DCS60), anti-K48-linkage specific polyubiquitin (D9D5), anti-phospho-histone H2AX (Ser139) (20E3), anti-phospho-ATM (Ser1981) (D6H9), anti-phospho-Chk1 (Ser345) (133D3), and anti-phospho-Chk2 (Thr68) (C13C1) were from Cell Signaling Technology (Beverly, MA, USA); anti-GAPDH and anti-HA-tag were from Bioworld Technology (St. Louis Park, MN, USA.).

Purified His-Uch37 and hRpn13 were dialysed extensively against phosphate buffer
to remove DTT and K48-linked tetraubiquitin (Boston Biochem) was dissolved in
the same buffer. Uch37, hRpn13 or hRpn13-Uch37 were incubated with RA190 or DMSO
rotating at 4 °C for 2 h, followed by addition of K48-linked
tetraubiquitin (final concentration of 1 μM K48-linked tetraubiquitin,
0.1% DMSO in all of reactions and final concentrations of 1 μM
Uch37, hRpn13, hRpn13-Uch37 or 20 μM RA190) for another 8 h at
37 °C. The reaction was quenched by adding SDS–PAGE loading
buffer (2% SDS, 10% glycerol, 2 M urea, 0.01%
Bromophenol Blue, 200 mM DTT) and heating at 80 °C for
8 min. Samples were subjected to SDS–PAGE and immunoblot
analysis.