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: rat spleen tissue lysate, Lane 2: rat thymus tissue lysate, Lane 3: rat brain tissue lysate, Lane 4: rat lung tissue lysate, Lane 5: mouse spleen tissue lysate, Lane 6: mouse brain tissue lysate, Lane 7: mouse HEPA1-6 cell lysate, Lane 8: mouse NIH3T3 cell lysate. 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 EEA1 at 0.5ug/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 1:10,000 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 EEA1 at approximately 170kD. The expected band size for EEA1 is at 162kD.