TNFα and the TNFα-induced second messenger ceramide activate members of the MAPK superfamily. (Panel A) Western blot of lysates from CLENDO cells treated for up to 60 min with TNFα (50 ng/ml) probed with antibody to phosphorylated ERK, p38, JNK and pan-ERK. Pan-ERK antibody, which detects all activated and inactivated ERK isoforms was used for normalization. (Panel B) Graphical representation of the percent change in expression of MAPK superfamily after densitometry of band developed in Western blots analysis as in Panel A and normalization to panERK. These data represent mean ± SEM of at least three independent experiments. * = P < 0.05. (Panel C) Treatment with ceramide, a product of TNFα treatment in CLENDO cells, for up to 60 min and Western blot detection of phosphorylated JNK isoforms. This Western blot is representative of similar results obtained in at least three separate experiments.