Western blot analysis of 045032 detecting the cleavage of endogenous mouse MDA5 as an indicator of apoptosis. Method: Cleavage of endogenous Helicard in murine A20 B lymphomas undergoing Fas-mediated apoptosis. A20 cells, either sensitive (S) or resistant (R) to FasL, were treated with 200 ng/ml of FasL for 4 hours and harvested at the indicated times. The cells were lysed and the cell extracts resolved by SDS-PAGE under reducing conditions. After transfer to nitrocellulose the membrane was incubated with anti-MDA5 (mouse), pAb at a dilution of 1:1'000 for 2 hours. Detection of MDA5 was performed using a peroxidase-conjugated antibody to rabbit Ig followed by a chemiluminescence detection system. Note: In non-stimulated A20S cells MDA5 has a molecular weight of 140kDa. Upon FasL-induced activation, cleavage products of 120, 41/45, 30 and 28kDa can be detected.