Figure 1. Western blot analysis of CRF/CRH using anti-CRF/CRH antibody (A00629-4). 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 SH-SY5Y whole cell lysates, Lane 2: human U-87MG whole cell lysates, Lane 3: human U251 whole cell lysates, Lane 4: rat brain tissue lysates, Lane 5: rat C6 whole cell lysates, Lane 6: mouse brain tissue lysates, Lane 7: mouse Neuro-2a 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-CRF/CRH antigen affinity purified polyclonal antibody (A00629-4) at 0.5 microg/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 CRF/CRH at approximately 25 kDa. The expected band size for CRF/CRH is at 21 kDa.