Cell culture and western blotting
HEK293T cells were purchased from American Type Culture Collection (Manassas,VA), cultured in Dulbecco’s modified Eagle’s media (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Korea, Seoul, Korea), and transfected with plasmid using Lipofectamine 2000 (Thermo Fisher Scientific Korea) according to the manufacturer’s instructions. Subsequently, the cells were lysed with M-PER mammalian protein extraction reagent (Thermo Fisher Scientific Korea) at 48 h post-transfection, and processed for Western blotting as described previously [27]. The antibodies used were anti-FLAG antibody (1:2000, Sigma-Aldrich, catalog number F1804), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (1:2000, Trevigen, Gaithersburg, MD, 2275-PC-100), HRP-conjugated goat anti-mouse antibody (1:4000, Santa Cruz Biotechnology, Dallas, TX, sc-2005), and HRP-conjugated goat anti-rabbit antibody (1:4000, Santa Cruz Biotechnology, sc-2004). Band intensity on the Western blots was analyzed using ImageJ.