Controlled processing The r and analyzed NO concentrations, as described below. After 30 min PFF or controlled The treatment the cells were lysed for b catenin quantification and analysis of levels of Akt phosphorylation LY2228820 and the expression of target genes b catenin. Nitric oxide. NO production as nitrite accumulation in the conditioned medium using the Griess reagent, measured as described above. b catenin quantification. The entire B catenin available to determine concentration in cell lysates, was used a total of b catenin enzyme immunoassay kit. Immediately after treatment PFF MLO Y4 cells were washed with PBS, and cell lysate was with RIPA buffer cell lysis with 0.5 lL / ml protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride collected.
b catenin quantification was carried out according to claim manufacturer’s protocol. Detection PI3-kinase of Akt phosphorylation. For the infrared detection of phosphorylated and unphosphorylated Akt, the blots were incubated with anti-phospho-specific antibodies Body proteins Antique Body, the specific protein Pan and monoclonal mouse anti-actin b prime Ren Antique rpern Overnight at 4 C in TBS Tween 20 0.05% incubated with 1% BSA as the blocking buffer by incubation with labeled secondary Ren Antique body IR for 60 min erg complements. The blots were imaged with Odyssey infrared imager at 700 nm and 800 canals le, each in a single scan resolution and high at 169 lm. Two-color detection were prime Re Antique Body of various species h You. Secondary Re Antique Body were of the same species, h You. Analysis of gene expression.
The gene expression of b catenin target genes connexin 43, CD44, cyclin D1, c, and June, using real-time PCR as described above. Performance of gene expression were normalized mouse GAPDH. The statistical analysis. The data were collected from four independent Get ngigen experiments. For statistical analysis, the PFF treated contr The ratios The overall concentrations and b-catenin gene expression of target genes were bcatenin calculated. The differences between the groups were students, two ST-test for paired comparisons with tail or with students,’s two-tailed t-test single group tested and compared to an average first Differences were considered significant if p 0.05. Results To determine whether mechanical stress by PFF to assess b-catenin in Module MLO Y4 osteocytes, the cells were treated with PFF or static culture conditions.
Application of PFF for 30 min at MLO Y4 osteocytes not Born in apparent consumption Used changes in cell shape or alignment of the cells in a specific orientation. No cells by treatment of the fluid flow have been removed, as assessed by visual inspection of cultures beforeAnd after treatment PFF. Three thirty minutes PFF treatment was sufficient to significantly regulate total of b catenin protein by the 1.7-fold as compared to contr The static, indicating that the fluid shear stress inhibits b catenin degradation in MLO Y4 osteocytes. To test whether the NO production unit was a total of b catenin content PFF-induced, were MLO Y4 osteocytes subjected to 30 min in the presence of PFF inhibitor, NO NAME L. PFF obtained Ht fa Is significant NO production at 5 and 10 min of 2.3 and 1.9 times, respectively. The PFF-induced NO production was in the presence of the name L, which also inhibited the PFF-induced increase in the total concentration of b catenin abolished, suggesting that PFF-induced NO