1% FBS DMEM F12 medium and then subjected to serum sti mulation for 24 h. The effects on cell growth were measured by 3 2,5 diphenyltetrazolium bromide and Thymi dine uptake assay. For studies with inhibitors, A549 cells were rendered quiescence for 24 h in 0. 1% FBS DMEM F12 medium and pretreated with U 0126 or SB 203580 for 30 min prior to serum stimulation for 12 h and selleck chemicals 24 h. The effects on cell growth were measured by Thymidine uptake assay. Thymidine uptake assay Thymidine was used at a concentration 0. 1 uCi per well. The Thymidine content of the cell lysates was determined by a scintillation counter and the values were expressed as counts per minute number of cells. In addition, cell number was analyzed using the Casy 1 System, based on the Coulter Counter principle.

PDE activity assay The A549 cell protein was extracted with RIPA buffer and equalized to the same concentration for use. The reactions were per formed with 10 ug protein in 100 ul HEPES buffer at pH 7. 6 consisting of MgCl2, BSA and cGMP at 37 C for 15 min. The samples were boiled for 3 min, subsequently cooled for 5 min and incubated with 25 ul Crotalus atrox snake venom for 15 min at 37 C. After being chilled on ice, the sam ples were applied to QAE Sephadex A 25 mini chromatography columns and eluted with 1 ml ammonium formate. The elutes were collected in 2 ml scintillation solution and counted by a beta counter with CPM values. Each assay was performed in triplicate and repeated twice independently. Data are expressed as picomoles of cGMP per minute per milligram of pro tein.

cGMP enzyme immunoassay At the end of culture, cells were washed with PBS twice and lysed in 0. 1 M HCl at room temperature for 10 min. After centrifugation the supernatants were equal ized to the same protein concentration for use. 50 ul protein samples which were pre diluted to 0. 3 ug ml and standard solutions were incubated with 50 ul tracer and 50 ul antibody in darkness at 4 C overnight. After washing 5 times, plates were incubated with Ellmans solution for 90 120 min at room temperature with gen tle shaking. The plates were read at a wavelength of 405 nm and the concentration was calculated by the ready made Cayman EIA Double workbook. The standard curve was made as a plot of the %B B0 value vs concentration of a series of known standards using a linear and log axis.

Using the 4 parameter logistic equation obtained from the stan dard curve, the cGMP concentration of samples was determined and is given as nmol mg protein. Each sam ple was determined in duplicate and repeated twice. Statistical analysis All data Brefeldin_A were expressed as the means S. E. Data were compared using a two tailed Students t test, or a 1 way ANOVA with the Bonferronis post hoc test for studies with more than 2 groups. Statistical significance was assumed when P 0. 05.