Western blot analysis of Nucleostemin using Nucleostemin antibody (Product # PA5-143898). 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 µg of sample under reducing conditions. Lane 1: human Hela whole cell lysates; Lane 2: human K562 whole cell lysates; Lane 3: human PC-3 whole cell lysates; Lane 4: human Jurkat whole cell lysates; Lane 5: rat C6 whole cell lysates; Lane 6: 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-Nucleostemin/GNL3 antigen affinity purified polyclonal antibody (Product # PA5-143898) 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 1:5,000 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 Nucleostemin/GNL3 at approximately 65 kDa. The expected band size for Nucleostemin/GNL3 is at 65 kDa.