Renilla and firefly luciferase activities were measured using a Dual-Luciferase Assay Kit (Promega) following the supplier’s protocol on a VICTOR X3 Plate Reader with injectors (PerkinElmer). Before measurement of luciferase activity, cells were lysed with the 1X Passive Lysis Buffer (Promega). FL-normalized RL expression levels for reporter and control were used to calculate fold repression as the ratio of control to reporter-normalized RL values.
Western blotting analysis of indicated proteins was performed as described previously (Pillai et al., 2005 (link); Ghosh et al., 2015 (link)). Supplemental Table S3 lists antibodies used for Western blot and IP. Imaging of all Western blots was performed using an UVP BioImager 600 system equipped with VisionWorks Life Science software, version 6.80 (UVP). Densitometric analysis of Western blot protein band intensities was quantified using ImageJ software.
