The epitope of endogenous Smad2 protein is changed after PGE2 stimulation. A, Serum-starved HMC were stimulated with 1µM of PGE2 for the indicated time. The total cell lysates (TCL) were collected and endogenous Smad2 protein was examined by immunoblotting with the Smad2N antibody, raised against a peptide near the N-terminus of Smad2 protein. B, Total RNA was isolated from HMC, and subjected to RT-PCR for EP1, EP2, EP3, and EP4 mRNAs using specific primers. C, Serum-starved HMC were pretreated with selective antagonists of EPs, SC51322 (EP1), AH6809 (EP2), or GW627368X (EP4) for 30min followed by stimulation with PGE2 for 60min, and endogenous Smad2 was examined by immunoblotting with the Smad2N antibody. The lower panel shows the results of three independent experiments (means± s.e.m). * , p <0.001 versus control; #, p> 0.05 versus PGE2-treated group.