Expression of protease-activated receptors in HeLa cells.

(A) Northern blotting. Total RNA (10 μg) from the indicated cell lines was probed using specific probes for human PAR-1, -2, -3, and -4 and for GAPDH as a loading control. Only PAR-1 mRNA was detected in non–serum-starved and serum-starved HeLa cells. Human umbilical vein endothelial cells expressed PAR-1 and –2, and the megakaryocytic DAMI cell line expressed PAR-3 and -4. (B) Stimulation with tethered ligand peptides for PAR-1, -2, and -4. Serum-starved HeLa cells were incubated for 45 minutes in the presence of 20 nM Xa or thrombin (IIa) as controls or the PAR-1 peptides SFLLRN (5 μM) or TFLLRNPNDK (5 μM), the PAR-2 peptide SLIGRL (5 and 100 μM), or the PAR-4 peptide GYPGKV (5 and 200 μM), followed by nuclear extract preparation for EMSA using NF-κB–specific probes.