Apliskan

To evaluate inhibition of interferon-stimulated response element (ISRE) promoter activation, HEK293T cells (5 × 10⁴ cells/well, 96-well plate format, triplicates) were transiently co-transfected using a calcium phosphate mammalian transfection kit (Agilent Technologies, Santa Clara, CA, USA), with 10 ng/well of pCAGGS plasmids encoding the WT or mutant 1918 PA-X proteins, or an empty plasmid as a control, together with 20 ng/well of a simian virus 40 (SV40)-driven Renilla luciferase (Rluc) expression plasmid and 50 ng/well of a plasmid expressing Firefly luciferase (Fluc) under the control of the ISRE promoter (pISRE-Fluc) [31 (link)]. At 24 h p.t., cells were washed and infected at a multiplicity of infection (MOI) of 3, with the Sendai virus (SeV), Cantell strain, for ISRE promoter activation [31 (link)]. At 21 h post-infection (h p.i.), cells were lysed using passive lysis buffer (Promega, Madison, WI, USA). Luciferase expression in the cell lysates was determined using a dual-luciferase kit (Promega, Madison, WI, USA) according to the manufacturer’s guidelines. Measurements were recorded with a microplate reader (Apliskan, Thermo Scientific, Waltham, MA, USA).