293T cells were maintained in DME medium as described [67] . Various constructs were expressed by transient transfection, using a liposome-mediated transfection protocol (5-10 mg of DNA/ 20 ml of Lipofectamine-2000 per 10 cm dish; Invitrogen) as described [67] . Cells were analyzed between 24 and 48 h after transfection. C-terminal EYFP fusion proteins of DUBs were generated by subcloning from pcDNA3.1 into pEGFP-N1 (Clontech). NP40 lysates of USP14 EYFP and USP14 C114S-EYFP transfected 293T cells were prepared and incubated with activesite probes as described [24] . Due to the high similarity with GFP, EYFP fusion proteins can be detected with a polyclonal anti-GFP rabbit serum [68] . Immunoblotting was performed as published [67] .