phorylation. To ana lyze order inhibitor total p44 p42 or other load controls, such as PARP, the same membranes were incubated for 30 min in strip ing solution at 55 C, washed twice with TBS T, and then reprobed with a primary antibody against the indicated protein. Immunoprecipitation TIC were scraped in ice cold TNTE buffer containing 5% Triton 100 and a protease inhibitor cocktail, the lysate was centrifuged for 10 min at 14,000 rpm at 4 C, and the solu ble fraction was incubated overnight with 3 ul of anti P2Y6 antibody. After that, 50 ul of protein G agarose was added to the lysate and incubated for 1 h at room temperature. the agarose beads were washed 3 times with TNTE containing 1% Triton 100 and protease inhibitors, resuspended in Laemmli buffer, boiled for 5 min, and analyzed by Western blot.

Proliferation assay Cell proliferation was analyzed using thymidine incorporation. For this, cells were cultured in 48 well plates and after 48 h of culture, they were harvested and incubated for 24 h in serum free DMEM F12 media. then the culture medium was changed to DMEM F12 with 0. 1% fetal bovine serum containing the e perimental treatment. Then cultures were incubated for another 48 h, with the addition of 1 u Ci well of thymidine after the first 24 h. At the end of the incubation, each well was washed 3 times with 5% trichloroacetic acid, and then the cells were lysed by addition of 250 ul of boiling 250 mM NaOH, incubated 5 min, and transferred to vials contain ing 5 ml of scintillation liquid. Samples were counted in a scintillation counter. Statistical analysis All data are e pressed as mean S.

E. M. Statistical analy sis was performed using GraphPad Prism software. The means of two groups were compared using a Students t test. ANOVA was used to compare several groups, and differences were considered to be significant at p 0. 05. Results Theca cell identity and e pression of P2Y2, P2Y4, and P2Y6 receptors TIC were isolated, and their identity was confirmed by RT PCR amplification of cyp11A, cyp17A, and star tran scripts as specific markers for theca cells, and of FSH receptor transcripts as indicator of a possible con tamination with granulosa cells. the B actin transcript was used as a control housekeeping gene. The results showed that TIC cultures were positive for cyp11A, cyp17A, and star e pression, but they did not e press the FSH receptor, demonstrating that the isolated cells Dacomitinib were mainly of the thecal interstitial type and were essentially free of granulosa cells.

This conclusion was strengthened with data obtained in functional e periments. For this, TIC cells were stimu lated by 2 IU hCG or 1 ng ml FSH, and CREB phosphory lation was evaluated. It is well established that gonadotropin sellckchem receptors e ert their actions by coupling to G proteins, increasing cAMP synthesis that, in conse quence, promotes CREB phosphorylation. It was found that in TIC cultures, CREB protein phosphoryla tion was increased 6 fold by hCG stimula tion, whereas FSH did not in

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