Ab152163

To validate our findings across species, we cultured human cardiac fibroblasts isolated from ventricles of adult heart (HCF, PromoCell # C-12377) in fibroblast growth medium 3 (PromoCell) until P5. Confluent cells were starved overnight in SFM and incubated with p1159 as described above (Table 1). After incubation, cells were rinsed and either fixed for immunofluorescence or lysed for protein and/or RNA extraction. Protein lysates were obtained with 1x ice cold lysis buffer with 1x protease inhibitors (Abcam, ab152163), according to manufacturer’s instructions and quantified for immunoblotting.

Confluent six-well-plates of A549 cells were transfected with 2 μg of plasmid pEF-BOS-RIG-I-Flag (provided by J. Yount, Ohio State University College of Medicine). At 24 h post-transfection, cells were lysed in lysis buffer (Abcam, ab152163). Cell lysates were harvested after centrifugation at 13,000×g for 10 min and incubated with Anti-FLAG^® M2 magnetic beads (Sigma-Aldrich, M8823) at room temperature for 80 min. The mixture was then divided into 13 aliquots (150µl/tube). 12 aliquots were incubated with 2×10⁸ copies of virion RNA (with or without CIP treatment) or 2×10⁹ copies of hMPV mRNA respectively at 37°C for 1 h. Beads associated RNA:protein complex were washed in lysis buffer for three times, and total RNA was extracted from beads by TRizol reagent and quantified by real-time RT-PCR. The 13^th aliquot was washed and subjected to Western blot.