Western Blot Analysis of BRET Assay

A portion of the cells collected for the BRET assay was used for Western blotting. Protein extracts (40 μg) from the transfected HEK293T/17 cells were loaded to commercially made 4–12% Bolt gel, separated by voltage potential using Mini Gel Tank (Thermofisher, Waltham, MA, USA), and transferred to polyscreen polyvinylidene difluoride transfer membrane (PerkinElmer, Waltham, MA, USA) using iBlot™ 2 Gel Transfer Device (Thermo Fisher Scientific, Waltham, MA, USA). Ponceau stain was used to demonstrate the equal loading of proteins in each well. Respective proteins were revealed by anti-RGS9 antibody (Proteintech, Rosemont, IL, USA, 17970-1-AP), anti-Gβ5 ATDG antibody [51 (link)], anti-R7BP antibody [29 (link)], and anti-GAPDH antibody (Millipore, Burlington, MA, USA, MAB374). Anti-Gβ5 antibody and anti-R7BP antibody were gifts from Dr. William Simonds (NIDDK, National Institutes of Health, Bethesda, MD, USA).