Western blot analysis of S6K1/RPS6KB1 using anti-S6K1/RPS6KB1 antibody (A01475-2). 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 30 ug of sample under reducing conditions. Lane 1: human MOLT4 whole cell lysates, Lane 2: human U20S whole cell lysates, Lane 3: human HL-60 whole cell lysates, Lane 4: human U251 whole cell lysates, Lane 5: human 293T whole cell lysates, Lane 6: human placenta tissue lysates, Lane 7: mouse NIH/3T3 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-S6K1/RPS6KB1 antigen affinity purified polyclonal antibody (Catalog # A01475-2) 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 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for S6K1/RPS6KB1 at approximately 59 kDa. The expected band size for S6K1/RPS6KB1 is at 59 kDa.