Detergent insoluble material was removed from the cell suspension by centrifugation at 12,000 g for 30 min. Proteins content was quantified using Bradford method. Aliquots of 30 ug supernatant proteins selleck bio from the differ ent samples were resolved by SDS PAGE. Electropho resed proteins were transferred to nitrocellulose membrane as described. The Inhibitors,Modulators,Libraries membranes were incubated with specific antibodies and then incubated with HRP coniugated anti species specific secondary antibodies. Immunoreactive bands were visualized by an enhanced chemiluminescence method The membrane was stripped and reprobed with an antibody calnexin to confirm equal protein loading per sample. Quantitative measurement of immunoreactive bands was performed by densitometric analysis using the Scion image software.

Data Inhibitors,Modulators,Libraries were then presented as fold change of the control. Immunofluorescence analysis For indirect immunofluorescence, C2C12 cells were fixed Inhibitors,Modulators,Libraries in 4% paraformaldehyde, permeabilized with 0. 2% Triton X 100, and blocked with PBS containing 1% bo vine serum albumin. Cells were then immunostained with specific antibodies rhodamine conjugated and nuclei re vealed with DAPI staining. Cells were observed using fluorescence Leica DM IRE2 microscopy and Nikon Eclipse 50I microscopy and images of myotubes were captured using respectively IM50 software and Nis Elements D 4. 00 software for size comparison. Data were displayed and analyzed using Adobe Photoshop CS4. For myotubes length and diameter size, the average measurement on each slide was generated from approxi mately 150 myotubes.

10 fields were randomly chosen and all MyHC positive multinucleated cells containing at least 3 nuclei in each field were measured. The data were then converted to percentage increase of the con trol. To quantify the differentiation and fusion of C2C12 cells after treatments, we calculated the fusion index as the average number Inhibitors,Modulators,Libraries of nuclei in of MyHC positive multinucleated cells above total nuclei. In the same way, the data were then converted to percentage increase of the control. Statistical analysis All experiments were performed three times. For array, immunoblotting and Immunofluorescence Inhibitors,Modulators,Libraries analysis, stat istical evaluations were performed by t test. Data are presented as the mean SD. Results were considered statistically significant if p 0. 05. Results Proliferative phase In proliferative phase, we investigated selleck chemicals MRFs protein syn thesis and morphologic features in C2C12 cells after ex posure to 0. 1 or 25 uM of RSV for different time periods. We used a control in which RSV was not added to the medium. We first examined RSV action on C2C12 proliferation rate. Every day, growth time and morphologic feature changes of C2C12 were evaluated.