RT PCR of Glyceraldehyde 3 Phosphate Dehydrogenase, a housekeeping gene, served as a control. Primers used in this study were as follows Two pairs of primers were added in the action tube for RT PCR. The PCR cycle conditions were as fol lows 2 minutes read more at 95 C, 30 seconds at 95 C, 35 seconds at 60 C, 40 cycles of 15 seconds at 95 C and 30 seconds at 60 C. Each PCR procedure included a negative control reaction without a template. Western blot analysis The cells were washed three times with ice cold PBS and lysed in RIPA lysis buffer. The lysates with Sodium dodecyl sulphate buffer were boiled for 5 minutes and separated by 10% SDS polyacrylamide gel electrophoresis. They were then transferred to a 0.

45 um polyvinylidene difluoride membrane and incubated with RANK antibodies at a dilution of 13000 and Horseradish peroxidase conjugated goat anti rabbit antibody at a dilution of 16000. The HRP substrate was observed on the PVDF membrane. After three washes, the PVDF membrane was incubated Inhibitors,Modulators,Libraries with B actin anti body and HRP conjugated antibody. The HRP substrate was observed again. For densitometric analyses, Inhibitors,Modulators,Libraries blots were scanned and quantified using Quan tity One analysis software, and the results were expressed as the percentages of B actin immunoreactivity. In vitro osteoclastogenesis assays and bone resorption assay BMMs or retrovirally infected BMMs were cultured in 24 well tissue culture Inhibitors,Modulators,Libraries plates in DMEM containing 10% FBS in the presence of 30 ngml M CSF and 100 ngml RANKL. Osteo clastogenesis began on the sixth day and the cultures were stained for tartrate resistant acid phosphatase on the ninth day using a kit in accordance with the manufacturer`s instructions.

TRAP positive cells appeared red to purple. Only TRAP positive cells with more than three nuclei were counted optically. The values Inhibitors,Modulators,Libraries were expressed as means and standard deviations of triplicate cultures. Bone resorption assays were performed using osteoclasts gener ated on mice bone slices from infected or uninfected BMMs as described Inhibitors,Modulators,Libraries previously. The bone slices were har vested on the ninth day. Osteoclastic resorption was quantified by measuring the area of resorption pits with digital image analysis software. The percentage of resorption was cal culated by dividing the resorbed area by the total area of the imaged bone slice surface. Statistical analysis Data are expressed as mean standard deviation.

The statistical significance AZD9291 astrazeneca of differences between the experi mental and the control groups was evaluated by inde pendent Students t test. Differences between the groups of the optimal shRANK selection were analyzed using analysis of variance with SPSSW 16. 0 software. P 0. 05 was considered to be statistically significant. Results and discussion As shown in Figure 1, nearly all cultured BMMs expressed the monocytemacrophage surface markers CD14 and CD34.