Deubiquitinating Enzymes: Expression and Mutation

Breast cancer cells, MCF7, lung cancer cells, A549, and HEK 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (#12800-017, Gibco), containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (P/S). The cells were maintained in a 5% CO₂ incubator at 37°C. Various full-length human DUB plasmids were cloned into the pDEST-CMV6 vector, and the plasmids are as follows: ubiquitin-specific peptidase 3 (USP3), USP21, USP22, USP25, USP28, USP28, USP35, USP36, USP39, UCH-L1, UCH-L5, and BAP1. To generate catalytic residue mutations, site-directed mutagenesis PCR (Polymerase Chain Reaction) was performed using the site-directed mutagenesis kit (#A14604, Invitrogen) according to the manufacturer’s protocol. The plasmids were as follows: USP21 C211A, USP35 C434A, and BAP1C91A. For DUBs overexpression, DUB plasmids were transfected using Lipofectamine 2000 reagent (#11668-019, Invitrogen), according to the manufacturer’s protocol.