Activation kinetics of endogenous MAP kinases in HUVEC after stimulation with TNF-, PMA, and arsenite. HUVEC were stimulated with 5 ng/mL TNF-, 20 ng/mL PMA, or 0.25 mmol/L arsenite for the time intervals indicated. Immunecomplex kinase assays were performed as described in Materials and Methods using 3pK K>M, GST-c-Jun(1-135), and myelin basic protein (MBP) as substrates for p38, JNK, and ERK, respectively. Protein loads were controlled in Western blot assays using appropriate antisera. On stimulation with PMA, a strong activation of ERK was detected, which persisted over 240 minutes (lower panel). Arsenite strongly induced p38 and JNK kinases with peak levels after 240 minutes (upper and middle panels).