The next day the culture medium was replaced with serum free medium and cells were stimulated with 10 8 M CRF for 3 hours. Cells were harvested, washed with PBS containing NaF and PMSF and incubated with promotion PFA 4% for 10 min. Cells were then washed with PBS and 0. 1% Triton X 100 was added for 15 minutes. Then, cells were incubated overnight with a monoclonal antibody against the phosphorylated form of FAK. Finally, cells were washed with PBS, stained with secondary anti mouse Ig FITC conjugated antibody raised in goat, for 1 hour and photographed with a Confocal Laser Scanning Microscope. Measurement of total prostaglandin production 2 105 cells were plated in 24 well plates and stimulated with CRF 10 8 for different time points. The supernatants of the cells were collected and stored at 80 C until ana Inhibitors,Modulators,Libraries lyzed.

The production of total prostaglandins was meas ured by the Prostaglandin Screening Inhibitors,Modulators,Libraries EIA Kit according to the manufacturers instructions. The assay is based on the competition between PGs and a PG acetylcholinesterase conjugate for a limited amount of PG antiserum. Western blotting analysis Western blot analysis of proteins for the detection of tubu lin, Cox 1 and Cox 2 was performed as previ ously described. Briefly, protein content in the lysates was measured by Bradford Assay. SDS PAGE sample load ing buffer was added in 10g of protein from each lysate and electrophoresed through a 12% SDS polyacrylamide gel. Protein was transferred to nitrocellulose membranes, using an LKB electroblot transfer system.

To detect protein levels, membranes were incu bated with the appropriate antibodies and then exposed to Kodak X omat AR films. A PC based Image Analysis was used to quantify the intensity of each band. Statistical analysis All values were expressed as the average Inhibitors,Modulators,Libraries Standard Error of data obtained from at least three independent experi ments. Comparison between groups was made using the ANOVA test and p 0. 05 was the signifi cance level. Results 1. Expression of CRF receptor subtypes in MCF7 cells To confirm that any biological effect of CRF in MCF7 cells occurred via the characterized Inhibitors,Modulators,Libraries CRF receptors we investi gated the expression of different CRF receptor subtypes. Expression of CRF1 has been previously reported in MCF7 cells. RNA from MCF7 cells was analyzed for the expression of CRF1a, Inhibitors,Modulators,Libraries CRF1b, CRF2a and CRF2c receptor subtypes by RT PCR.

Among these four subtypes, CRF1a mRNA was expressed in high levels while CRF2c mRNA was present at selleck chemical very low levels. The mRNAs of CRF1b, CRF2a were detected in human hippocampus but were not detected in MCF7 cells. 2. CRF affects apoptosis of MCF7 cells in a time dependent manner Evasion of apoptosis is a hallmark of cancer cells and is frequently associated with proliferation and invasiveness. It has been previously reported that CRF has anti proliferative effects on cancer cell lines such as Ishikawa endometrial carcinoma cells and in MCF7 stimulated by estrogens.