PLSCR1 does not affect the phosphorylation status of NP and does not stimulate the IFN pathways.

(A) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. Cell lysates were processed at 6 h and 8 h p.i., immunoprecipitated with a mouse anti-NP mAb, a mouse anti-p-Ser mAb, or a mouse anti-p-Tyr mAb, followed by western blotting with a rabbit anti-NP pAb to detect the level of total NP, serine-phosphorylated NP, and tyrosine-phosphorylated NP, respectively. (B) Replication of an NP-phosphorylation mutant virus in PLSCR1-overexpressing A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with wild-type WSN virus or the phosphorylation mutant S9A/Y10F at an MOI of 0.1. Supernatants were collected at the indicated timepoints and titrated for infectious virus by means of plaque assay on MDCK cells. (C) Expression of Mx1 protein in PLSCR1-overexpressing or control A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were grown in 12-well plates, and were left untreated or were treated with IFN-α for 24 h. The cell lysates were then subjected to western blotting with a rabbit anti-Mx1 pAb for the detection of Mx1 protein. GAPDH, detected by a rabbit anti-GAPDH pAb, served as a negative control. (D) Expression of the ISRE luciferase reporter gene in HEK293T cells transfected with the PLSCR1-expressing construct or empty vector. HEK293T cells were transfected with the ISRE-Luc reporter plasmid, pRL-TK control plasmid, and the pCAGGS-PLSCR1 or empty pCAGGS plasmid for 20 h. The overexpression of PLSCR1 was confirmed by western blotting with a rabbit anti-PLSCR1 pAb. The luciferase activity of the transfected cells was analyzed by using the Dual-Luciferase reporter assay. After normalization with co-transfected Renilla luciferase activity, the relative firefly luciferase activity of PLSCR1-overexpressing cells was expressed as the fold-induction of the ISRE firefly luciferase activity compared to cells transfected with empty pCAGGS vector. **, P < 0.01.