Ha k48

The expression plasmid encoding OTUB1-flag and OTUB1 truncation mutants were constructed by PCR and subsequent insertion of the corresponding fragment into GFP-N1 vector. The YAP-myc and YAP truncation mutants were constructed by PCR and subsequent insertion of the corresponding fragment into pcDNA3.1 vector. The HA-K48, K63, HA-Ub and TEAD-reporter plasmids were purchased from Addgene. The plvx-shRNA2-OTUB1, pCDH-EF1-OTUB1-flag, pCDH-EF1-YAP-flag and pCDH-EF1-YAP-S5A-flag were constructed into corresponding vector. To produce lentivirus, the expression vector transfected into HEK293 cells along with the packaging plasmid psPAX2 and the envelope plasmid pMD2.g using Lipofectamine 2000 (Invitrogen). The siRNA sequences were as follows: siOTUB1-1: 5′-GAC GGC AAC UGU UUC UAU C-3′, and siOTUB1-2: 5′-GAC GGA CUG UCA AGG AGU U-3′. The shRNA sequences were as follows: shOTUB1-1: GGA TCC GTT AAC TGT CTG GCC TAT GAT TCA AGA GAT CAT AGG CCA GAC AGT TAA TTT TTT GAA TTC and siOTUB1-2: GGA TCC GGA CGG ACT GTC AAG GAG TTT TCA AGA GAA ACT CCT TGA CAG TCC GTC TTT TTT GAA TTC.