Western blot analysis of APPL/APPL1 using APPL/APPL1 antibody (Product # MA5-49222). 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 50 µg of sample under reducing conditions. Lane 1: human HL-60 whole cell lysates; Lane 2: human THP-1 whole cell lysates; Lane 3: human PC-3 whole cell lysates; Lane 4: human Hela whole cell lysates; Lane 5: rat brain tissue lysates; Lane 6: mouse brain 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 mouse anti-APPL/APPL1 antigen affinity purified monoclonal antibody (Product # MA5-49222) 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-mouse 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 APPL/APPL1 at approximately 85KD. The expected band size for APPL/APPL1 is at 85KD.