The TM cells were maintained in serum-free medium for 18 hours before various treatments. To activate the p42/44 MAPK, the cells were pretreated with URB597 for 15 minutes, followed by treatment with PEA plus URB597 for 10 minutes. To study the effects of various antagonists or inhibitors, the cells were pretreated with URB597 plus these antagonists or inhibitors (SR141716A, SR144528, GW6471, or PD98059) for 15 minutes, followed by PEA plus URB597 treatment for 10 minutes. At the end of the incubation period, 150 μL of ice-cold lysis buffer containing 50 mM β-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 1 mM NaVO4, 1 mM dithiothreitol (DTT), and 1 μg/mL of a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) were added. The cell lysates were incubated on ice for 5 minutes and then transferred to microcentrifuge tubes. The lysates were clarified by centrifugation at 14,000g for 10 minutes, the supernatants were collected, and protein concentrations were measured (Bradford protein assay reagent; Bio-Rad, Hercules, CA). After boiling with 2× Laemmli sample buffer for 5 minutes, 40 μL of cell lysate (containing 25 μg of protein) was run on a 10% SDS–polyacrylamide gel. Subsequently, the proteins were transferred onto a nitrocellulose membrane, and the total p42/44 MAP kinase bands were detected by Western blot analysis by using a rabbit polyclonal anti-p42/44 MAP kinase antibody (Cell Signaling Technology, Beverly, MA). The levels of phosphorylated p42/44 MAPK were determined using a mouse monoclonal antibody against phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), according to procedures described by the manufacturer.