Renilla luciferase

LNCaP prostate tumor cells were stably transduced to express firefly luciferase under control of the ER stress transcriptional response element, as previously described [44 (link)]. As an internal control, these cells were also transduced to constitutively express Renilla luciferase (Thermo Fisher Scientific) under control of the cytomegalovirus promoter. Transduced cells were selected in medium containing 1 μg/mL puromycin (Life Technologies, Carlsbad, CA), and single-cell clones were either mock-irradiated (0 Gy) or exposed to 10 Gy ²²³Ra over a 96-h incubation period. Luciferase activity was quantified using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) at multiple time points after the start of treatment to monitor the progressive onset of the ER stress response pathway.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were conducted as previously described [26 (link), 27 (link)] with the following modifications. Briefly, whole cell lysates from plasmid-transfected HEK293T cells were harvested using Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA, USA) then separated using 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Antibodies against Renilla luciferase (Thermo Fisher Scientific), β-tubulin (Sigma-Aldrich) and anti-PRRSV-N monoclonal antibody generated in-house (clone No. 6D10) were used for specific protein detection. Detection of primary antibody binding to targets was conducted by incubation of membranes with goat anti-rabbit IgG-conjugated or anti-mouse IgG-conjugated horseradish peroxidase (Sigma-Aldrich) and visualized after addition of ECL chemiluminescence substrate (Bio-Rad Laboratories). The chemiluminescence signal was recorded digitally using a ChemiDoc MP imaging system (Bio-Rad Laboratories). Digital signal acquisition and densitometry analyses were conducted using ImageLab Software, Version 5.1 (Bio-Rad Laboratories).