HEK 293 cells were transfected with vectors expressing either wildtype (WT) HA-ERK8 or HA-ERK8 [D154A]. At 36 hr post-transfection, the cells were exposed for 10 or 30 mins to 1 mM hydrogen peroxide, and lysed. The lysates (60 μg) were denatured by SDS, subjected to SDS-PAGE, transferred to nitrocellulose membrane and blotted with the anti-ERK8 antibody at 0.5 μg/ml. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti-sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).