293T cells were co-transfected with pcDNA3.1/Myc-Egr3 plasmid and Egr3 siRNA. Cells transfected with empty plasmid or non-targeting siRNA served as negative controls. Cell lysates were prepared in RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1X Protease Inhibitor [Roche, Indianapolis, IN, USA]) and centrifuged at 13,000 rpm. The supernatant was subjected to bicinchoninic acid (BCA) assays (Thermo Scientific, Rockford, IL, USA) for quantitation. Fifty micrograms of protein were loaded per lane and separated in 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels. Western blotting was performed as previously described [2 (link)]. Anti-α-tubulin (Sigma-Aldrich, USA) and anti-Egr3 (sc-191; Santa Cruz Biotechnology, USA) antibodies were used at 1:2,000 and 1:500, respectively. Anti-Myc antibody (#631206; Clontech, USA) was used at 1:2,000. Horseradish peroxidase (HRP) -conjugated goat anti-rabbit and goat anti-mouse antibodies (GeneDEPOT, Barker, TX, USA) were used at 1:10,000. Super Signal West Femto ECL reagent (Thermo Scientific, USA) was used. Chemiluminescence signal was detected by LAS3000 (FUJIFILM, Tokyo, Japan).