WP1130 inhibits DUB activity and reduces Mcl-1 protein expression in myeloma cells. (A) Myeloma cells (as indicated) were treated with 5 μM WP1130 for 0, 2, or 4 hours before DUB activity was assessed by hemagglutinin-ubiquitin vinylsulfone (HA-UbVS) labeling. The labeled DUBs were detected by immunoblotting with anti-HA antibody. HA-labeled Usp9x is denoted. The protein lysates were also immunoblotted for total Ub, Usp9x, Mcl-1, and PARP. β-actin was used as a protein loading control. (B) Z138 and Mino cells were treated as noted before cell lysates were screened for Usp9x activity (HA-labeled Usp9x), Usp9x, Mcl-1, PARP, and actin protein level by blotting. (C) Myeloma cells were treated with WP1130 alone or were pretreated for 30 minutes with proteasome inhibitors MG-132 (5 µM) or bortezomib (Bz) (50 nM) prior to WP1130 treatment. Lysates were prepared after 4 hours and immunoblotted for Ub and Mcl-1. Equal protein load was confirmed by immunoblotting with β-actin.