The expression plasmid for HA-tagged PA-X was generated by PCR mutagenesis of the pCR3.1-PA-X vector (26 (link)) to insert tandem HA tags at the C terminus of the PA-X open reading frame to produce pCR3.1-PA-X-2HA vector. The expression vector for the Myc-tagged NS1 protein pCR3.1-NS1-myc is described in (30 (link)).
For testing the effects of NS1 on PA-X shutoff activity, 293 A cells were co-transfected with the pCR3.1-EGFP reporter vector described in (27 (link)) and the pCR3.1-PA-X-2HA and/or the pCR3.1-NS1-myc expression constructs using linear polyethylenimine (PEI, 23966–1, Polysciences, Warrington, PA) at the ratio of 500 ng total DNA to 1.5 µL of 1 mg/mL PEI per well of a 12-well cluster dish in Opti-MEM-I media (Thermo Fisher Scientific). In control transfections, the pCR3.1-PA-X-2HA and/or pCR3.1-NS1-myc expression vectors were substituted with the pCMV-LUC2CP/ARE control vector expressing firefly luciferase [a gift from Gideon Dreyfuss, Addgene plasmid #62857 (72 (link))]. Protein expression was analyzed at 24 h post-transfection using western blotting.
For testing the effects of NS1 on PA-X shutoff activity, 293 A cells were co-transfected with the pCR3.1-EGFP reporter vector described in (27 (link)) and the pCR3.1-PA-X-2HA and/or the pCR3.1-NS1-myc expression constructs using linear polyethylenimine (PEI, 23966–1, Polysciences, Warrington, PA) at the ratio of 500 ng total DNA to 1.5 µL of 1 mg/mL PEI per well of a 12-well cluster dish in Opti-MEM-I media (Thermo Fisher Scientific). In control transfections, the pCR3.1-PA-X-2HA and/or pCR3.1-NS1-myc expression vectors were substituted with the pCMV-LUC2CP/ARE control vector expressing firefly luciferase [a gift from Gideon Dreyfuss, Addgene plasmid #62857 (72 (link))]. Protein expression was analyzed at 24 h post-transfection using western blotting.
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