PARP9 Binding to Reovirus dsRNA

Aliquots of cell-free PARP9 recombinant protein (0.5, 1, 5 μg) were mixed with 1 pmol of biotinylated reovirus dsRNA Reo1198 (Reo1198) or unlabeled viral dsRNA Reo1198 (1, 5, 20 μg) for competitor assay, and incubated at room temperature for 1 h. Controls included, protein only and biotinylated-Reo1198 only reactions. After incubation for 1 h, proteins and biotinylated Reo1198 dsRNA were crosslinked with 1% formaldehyde for 30 min on ice. Crosslinking was quenched by adding glycine to a final concentration of 125 mM. Reactions were subjected to gel electrophoresis (Novex TBE-Urea 10% Gels, Invitrogen, Carlsbad, CA, USA) and transferred to nylon membranes (ThermoFisher Scientific). Biotin-labeled dsRNA was detected using the LightShift Chemiluminescent RNA EMSA Kit (ThermoFisher Scientific)^{63 (link),66 (link)}.