pLVSIN nFLAG3 human PP2Ac, PME-1, and PP6c were previously described (7 , 43 (link)). pLVSIN nFLAG3 human PP2Ac S24A, S30A, T40A, S43A, T78A, S93A, T219A, S225A/T227A, T235A, S285A, and T301A, pcDNA3 nFLAG2 PP2Ac and PP4c, pLVmCherry for non-target shRNA (shNT) and PME-1 targeting shRNA (shPME-1) were generated using InFusion HD Cloning Kit (Takara Bio). The sequences of shRNA are as follows: shNT, 5′-CAACAAGATGAGAGCACCA-3′, shPME-1#1, 5′-GGGCGATACATCTGAGTTCA-3′.
To produce lentiviruses, pLVSIN, a packaging plasmid (psPAX2), and a coat protein plasmid expressing vesicular stomatitis virus G protein (pMD2.G) were diluted in Opti-MEM containing Polyethylenimine MAX (PEI, Polysciences). The mixture was added to Lenti-X 293T cells (Takara Bio) Medium was replaced with fresh medium after 8 h, and cells were cultured for 48 h. The viral supernatants were filtered using a 2.2 μm filter (Millipore) and added to cells.
To produce lentiviruses, pLVSIN, a packaging plasmid (psPAX2), and a coat protein plasmid expressing vesicular stomatitis virus G protein (pMD2.G) were diluted in Opti-MEM containing Polyethylenimine MAX (PEI, Polysciences). The mixture was added to Lenti-X 293T cells (Takara Bio) Medium was replaced with fresh medium after 8 h, and cells were cultured for 48 h. The viral supernatants were filtered using a 2.2 μm filter (Millipore) and added to cells.
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