A set of standards of known concentrations for see more IGF-1 and HGF were utilized to construct standard curves by plotting the net absorbance values of the standards against their respective protein concentrations. By applying a four part parameter curve using MikroWin microplate data reduction software (Microtek Lab Systems, Germany), the free IGF-1 and HGF concentrations in the serum samples were calculated. The overall intra-assay percent coefficient of variation was 4.9% and 3.3% for IGF-1 and HGF, respectively. Skeletal muscle phosphorylated c-met content and MRF ELISAs Approximately 20 mg of each muscle sample was weighed and subsequently homogenized using a commercial

cell extraction buffer (Biosource, Camarillo, CA) and a tissue Selleck PD173074 homogenizer. The cell extraction buffer was supplemented with 1 mM phenylmethanesulphonylfluoride selleck chemical (PMSF) and a protease inhibitor

cocktail (Sigma Chemical Company, St. Louis, MO) with broad specificity for the inhibition of serine, cysteine, and metallo-proteases. Muscle homogenate samples were analyzed for phosphorylated c-met (Tyr1230/Tyr1234/Tyr1235) using a phosphoELISA kit (Millipore, Billerica, MA). This sensitivity of this particular assay is reported to be 0.78 U/ml. The absorbances, which are directly proportional to the concentration of c-met in the samples, were measured at 450 nm with a microplate reader (Wallac Victor 1420, Perkin Elmer, Boston MA). pheromone A set of standards of known concentrations for c-met were utilized to construct standard curves by plotting the net absorbance values of the standards against their respective protein concentrations. By applying a four part parameter curve using MikroWin microplate data reduction software (Microtek Lab Systems, Germany), the c-met concentrations in the muscle samples were appropriately calculated. The overall intra-assay percent coefficient of variation was 6.89% The muscle protein expression of the MRFs was assessed through the use of ELISAs. Polyclonal antibodies specific for Myo-D, myogenin, MRF-4, and myf5

(where their target specificities had been verified by Western blotting) were purchased from Santa Cruz Biotech (Santa Cruz, CA). Initially, the antibodies were diluted to 1 μg/ml in coating buffer (Na2CO3, NaHCO3, and ddH2O, pH 9.6) and allowed to incubate at room temperature overnight. Following incubation, the plates were washed (1× phosphate buffered saline, Tween-20), blocked (10× phosphate buffered saline, bovine serum albumin, ddH2O), washed, and then incubated with a secondary antibody (IgG conjugated to HRP) diluted to 1 μg/ml in dilution buffer (10× phosphate buffered saline, Tween-20, bovine serum albumin, ddH2O). After washing, a stabilized TMB chromogen was added and the plates were covered and placed in the dark for the last 30-min prior to being stopped with 0.2 M sulphuric acid.