PEITC inhibits endogenous MEKK1 and downstream signaling to SAPK. LNCaP cells were incubated with PEITC at the indicated concentrations for 15 minutes at 37°C. Hyperosmotic shock was induced by addition of sorbitol to a final concentration of 300 mM and cells were incubated for an additional 15 min. Lysates were prepared, MEKK1 and SAPK were isolated by immunoprecipitation and their activity was measured by in vitro kinase assay with SEK-KR (MEKK1, upper panel) or jun (SAPK, lower panel) as substrate. Activation of endogenous MEKK1 by hyperosmotic shock was dose dependently inhibited by PEITC pretreatment. The inhibition of MEKK1 exactly paralleled inhibition of SAPK.