Tissue culture reagents and fetal calf serum were purchased from ICN Biochemicals Inc. (Costa Mesa, CA). Human PGHS-2 cDNA was donated by Dr. Gary O’Neill (Merck Frosst Canada Inc., Pointe-Claire, PQ, Canada). The polyclonal antibodies to cPLA2, PGHS-1, and PGHS-2 were obtained from Dr. J. Grassi (CEA, Saclay, France). The monoclonal antibody to PGI synthase was from Oxford Biomedical Research Inc. (Oxford, MI); [a-32P]dCTP (> 3.000 Ci/mmol) was purchased from Dupont (Boston, MA). All other chemicals were supplied by Sigma Chemical Co. (St. Louis, MO). Buy Advair Diskus Online
Cell Culture and Treatment
Bovine uteri were collected at local slaughterhouses, and the stage of the estrous cycle at which the uteri were collected (midluteal phase) was estimated by examining the ovarian morphology. Myocytes from bovine myometrium were cultured in RPMI 1640 supplemented with 10% fetal bovine serum as previously described. When cell cultures reached confluency (Days 7-10 after plating), the culture medium was replaced with fresh medium without serum. The cells were then cultured in the absence or presence of 100 nM phorbol 12-myristate (PMA) to stimulate production of PGs.