Figure 1. Western blot analysis of Phospho-RIP3 (S232) using anti-Phospho-RIP3 (S232) antibody (M00202S232). 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: rat spleen tissue lysates, Lane 2: rat liver tissue lysates, Lane 3: rat heart tissue lysates, Lane 4: rat L6 whole cell lysates, Lane 5: mouse spleen tissue lysates, Lane 6: mouse liver tissue lysates, Lane 7: mouse heart tissue lysates, Lane 8: mouse L929 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-Phospho-RIP3 (S232) antigen affinity purified monoclonal antibody (Catalog # M00202S232) at 1:500 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 1:500 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 Phospho-RIP3 (S232) at approximately 50 kDa. The expected band size for Phospho-RIP3 (S232) is at 50 kDa.