Additional file 3: Fig. S3. of G45R mutation in the nonstructural protein 1 of A/Puerto Rico/8/1934 (H1N1) enhances viral replication independent of dsRNA-binding activity and type I interferon biology

Western blot analysis of STAT1, pSTAT1 and NS1 protein. A549 cells were infected with rX31 encoding WT and G45R/NS1 viruses at an MOI of 2. At 8 h post infection, cell lysates were clarified by centrifugation at 13,000 rpm for 15 min. The lysates were mixed in Laemmli sample buffer (Bio-Rad) and denatured at 95 °C for 5 mins prior to perform SDS-PAGE gel electrophoresis. Immunoblots were probed for STAT1 (purified rabbit anti-Stat1 N-terminus, BD Transduction Laboratories), pSTAT1 (purified mouse anti-Stat1 pY701 BD Transduction Laboratories), NS1 (polyclonal rabbit anti-NS1; Thermoscientific) and actin (purified mouse anti-actin Ab-5; BD Transduction Laboratories). Antibodies were detected by incubation with goat anti-rabbit (GE healthcare) or goat anti-mouse HRP-linked antibody (Cell signaling). The immunoblots were visualized by using ChemiDoc XRS imager (Bio-Rad). Band intensity of proteins was quantified by using Image Lab version 5.0 (Bio-Rad). (TIFF 2319 kb)