SMYD5 Antibody / SET and MYND domain-containing protein 5 (FY12912)

Western blot analysis of SMYD5 using anti-SMYD5 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human HepG2 whole cell lysates, Lane 2: human whole cell lysates, Lane 3: human Caco-2 whole cell lysates, Lane 4: human U2OS whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-SMYD5 antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using an ECL Plus Western Blotting Substrate. A strong ~47 kDa band is detected with additional species at ~60-65 kDa and ~70 kDa. The higher bands are consistent with post-translationally modified SMYD5, particularly ubiquitinated and SUMOylated forms, which add ~8-12 kDa per moiety and yield discrete higher-migrating species, as reported for ubiquitin/SUMO mobility shifts in western blot analyses.