Western blot analysis of RSK3 using anti-RSK3 antibody. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. Lane 1: human placenta tissue lysates,Lane 2: human Hela whole cell lysate,Lane 3: human PC-3 whole cell lysate,Lane 4: human A431 whole cell lysate,Lane 5: human K562 whole cell lysate,Lane 6: human PANC-1 whole cell lysate,Lane 7: rat testis tissue lysates,Lane 8: mouse testis tissue lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-RSK3 antigen affinity purified polyclonal antibody at 0.5 µg/mL overnight at 4°C, 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 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for RSK3 at approximately 90-100KD. The expected band size for RSK3 is at 83KD.