It is important to understand here that we are not comparing the tox icity of d

These cells were maintained at 37 ABT-737 価格 C with 5% CO2 and subcultured at approximately 60% to 70% confluence in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum. To assess the im pact of osmotic shock on the activation of ERK1 2, cells were treated with D sorbitol for 1 h and pro teins were harvested in RIPA extraction buffer as previously described. Quantitative real time reverse transcription polymerase chain reaction Total RNA was extracted using the RNeasy isolation kit. Total RNA was used to synthesize cDNA using SuperScript First strand Synthesis System according to the manufacturers instruc tions. For each replicate in each experiment, RNA from tissue samples of different animals was used. Primers were designed corresponding to mouse RNA se quences using Primer3.

Real time quantitative AEB071 構造 RT PCR reactions contained HotStart IT SYBR green qPCR Master Mix, 200 nM of each primer and 0. 2 uL of template in a reaction volume of 25 uL. Amplification was carried out using the ABI 7300 Real time PCR System. Relative levels of mRNA expression were calculated using the CT method and individual expression values were normal ized by comparison to Gapdh mRNA. Protein extraction and immunoblotting Skeletal muscle was homogenized in RIPA extraction buffer as previously de scribed. Extracted proteins were separated by SDS polyacrylamide gel electrophoresis, transferred to nitro cellulose membranes, and blotted with primary anti bodies against ERK1 2 and phosphorylated ERK1 2. Secondary antibodies were horse radish peroxidate conjugated.

Recognized proteins were visualized by enhanced chemiluminescence. To quantify results, the immunoblots AG-014699 溶解度 were scanned and band densities calculated using ImageJ64. Signals obtained for phosphorylated ERK1 2 were normalized to those for total ERK1 2. Serum biochemistry Serum was separated from mouse blood and stored at 80 C for 3 to 9 months until analyzed. Creatine phosphokinase and aspartate aminotransferase activities were measured using an Analyst III Analyzer in the Comparative Pathology Laboratory at Columbia University Medical Center. CPK and AST activities have been shown to be stable in rodent serum stored for up to 360 days at 70 C. Limb grip strength measurements LmnaH222P H222P mice treated with DMSO or selumetinib were subjected to limb grip strength testing using a hori zontally positioned grip strength meter.

Mice were lowered by the tail towards the grid on the apparatus. Upon grasping the grid with their limbs, mice were pulled backward in the horizontal plane. The procedure was re peated consecutively three times and the peak tension of the three pulls was recorded as the grip strength value. Each animal was subjected to a total of two serial trials of three pulls each with 20 s of rest in between. Statistics Values for real time quantitative RT PCR, scanned im munoblots, internalized nuclei, serum CPK and AST ac tivities, and grip strength were compared using an unpaired Student t tests. Values for Ferets diameter were compared using two way ANOVA. Statistical analyses were performed using Prism. Results and discussion Dystrophic skeletal muscle pathology in LmnaH222P H222P mice Arimura et al.