Luciferase Assay for PKR Plasmid Activity

Luciferase assays were performed following the protocol described previously [45 (link)]. Briefly, HeLa-PKR^kd cells (5 × 10⁴ cells/well) were seeded in 24-well plates a day before the transfection. For each transfection reaction mix, 0.05 µg of firefly luciferase (Promega) and 0.2 µg of each of the indicated PKR plasmids were co-transfected with 0.8 µg of either E3L, CRV157, CRV157-K48A/K49A, CRV157 del C, or CRV157 del N plasmids using GenJet (SignaGen Laboratories, Frederick, MD), with transfection reagent to DNA ratios of 2:1. For the titration experiments, the indicated concentrations of CRV157 or CRV157 del C plasmids were co-transfected. Each transfection was performed in triplicate, and plasmid DNA concentrations for each transfection mix were kept constant by adding an appropriate amount of pSG5 vector plasmid where necessary. After 48 h, whole-cell lysates were extracted using mammalian lysis buffer (GoldBio, St Louis, MO), USA, followed by the addition of luciferin (Promega, Madison, WI, USA). Luciferase activity was determined in a Glowmax luminometer (Promega). Each experiment was conducted at least three times, and representative experiments are shown.