Figure 1. Western blot analysis of EIF4E using anti-EIF4E antibody (M00135). 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 293T whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human HepG2 whole cell lysates, Lane 4: human A549 whole cell lysates, Lane 5: rat RH35 whole cell lysates, Lane 6: rat PC-12 whole cell lysates, Lane 7: mouse HEPA1-2 whole cell lysates, Lane 8: 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-EIF4E antigen affinity purified monoclonal antibody (Catalog # M00135) at 1:100 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 EIF4E at approximately 25 kDa. The expected band size for EIF4E is at 25 kDa.