Geneticin g418

Flag-CRIP1, empty vector and shRNA plasmids were purchased from Youbio Biological Technology (Changsha, China). The full-length coding sequence of CRIP1 was inserted into pCDH-EF1-MCS-T2A-Puro. shRNAs targeting CRIP1, PSME4 and USP7 were purchased from OBiO Technology (Shanghai, China). Endotoxin-free transfection-grade plasmid DNA was amplified in E. coli DH5α Competent Cells (Sangon Biotech, Shanghai, China) and purified with the EndoFree Midi Plasmid Kit (TIANGEN, Beijing, China). Lentiviruses were constructed using psPAX2 and pMD2G and transfected into HEK293T cells with Lipofectamine 3000 (Invitrogen, USA), harvested after 48 or 72 h, and concentrated through ultracentrifugation (20,000×g, 2.5 h, 4 °C). The lentivirus was added to 1 $\times$ 10⁶ cells along with 8 mg/mL polybrene in a 12-well plate, and stable cells were selected after 72 h using puromycin (60210es25, Yeasen, China) and geneticin (G418; Selleck, USA). The levels of CRIP1 were confirmed using RT-qPCR and western blotting. shRNA sequences are listed as follows: CRIP1-shRNA (5′-AGCACGAAGGCAAACCCTACT-3′); PSME4-shRNA (5′-GCAACTAGTAAATCTCTTTGC-3′); and USP7-shRNA (5′-GTGTCCTATATCCAGTGTAAA-3′).