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Assay of creatine kinase activity Cells were scraped with 500 ul of ice cold lysis buffer containing 20 mM Tris HCl, 100 mM NaCl, 1% Triton and protease inhibitor cocktail. Lysates were kept on ice during 15 minutes and cleared by centrifuga tion at 13,000 g for 15 minutes. The creatine kinase activ ity assay was performed by using a CK NAC LD B kit AP24534 価格 from Sobioda, which allows to monitor at 340 nm the kinetics of NADPH formation. The assay was performed in 96 well plates, with 4 uL of sample and 100 uL of reagent per well, for 20 minutes at 30 C. ELISA of myosin heavy chain Cells were scraped in 300 uL ice cold RIPA buffer, vortexed and centrifuged at 10,000 g for 10 minutes. The assay was carried out in 96 well plates on 50 uL of 1 50 diluted samples.<br><br> The wells were evaporated to dry ness overnight at 37 C and washed twice with cold PBS, using an automatic plate washer. Unspecific binding sites were saturated with 100 uL of 0. 3% BSA in PBS for 30 minutes at 37 C. Samples were then incu bated with 50 uL MF 20 antibody diluted 1 100 in PBS, for 1 hour at 37 AT7519 溶解度 C. After a new washing step in 0. 2% Tween 20 in PBS, incubation with 50 uL of secondary HRP conjugated anti mouse IgG antibody diluted 1 3000 was performed for 1 hour at 37 C. Plates were washed 5 times, 50 uL of TMB substrate were added to each well, and 50 uL 0. 5 N H2SO4 were added after 5 min to stop color reaction. Optical Density was read at 450 nm. A standard curve was obtained with purified myosin heavy chain. Western blotting Cells were lyzed as for CK assay, in the presence of 10 mM sodium pyrophosphate, 10 mM glycero phosphate, 50 mM NaF, 1.<br><br> 5 mM Na3VO4. Cell lysates were analyzed by SDSPAGE, and proteins were transferred onto PVDF membranes buy Alisertib blocked with 5% BSA in Tris buffered saline0. 1% Tween 20, and incu bated with appropriate antibodies following manufac turers recommendations. Immunoblots were revealed with ECL detection system and quantified with Image J software. SDS PAGE was performed using 10% polyacrylamide gels for S6K1 and Akt. In the case of PLDs, samples were subjected to SDS PAGE on 8% polyacrylamide gels, in the presence of 4 M urea. In vivo experiments 5 week old male BALBc mice were obtained from Charles River France. Animals were housed in the ani mal facility under standard conditions.<br><br> Adenovirus en coding PLD1 were injected in the right gastrocnemius, the left gastrocne mius being injected with the same amount of control GFP encoding adenovirus. The animals were sacrificed 10 days post injection, gastrocnemius muscles were dis sected from both hind limbs, frozen in liquid N2 cooled isopentane and stored at −80 C for either histological or molecular analyses. Muscle cryo sections were stained with Hematoxylin Eosin, and fibre cross sec tional areas were mea sured by using NIH Image J software. Alternatively, sections from the PLD1 injected muscles were immuno labeled for laminin and for HA tag, to respectively deter mine fibre outline and detect PLD1 expressing fibres. Fibre CSA was determined as above. Mice were treated in strict accordance to the guide lines of the Institutional Animal Care and Use Commit tee and to relevant national and European legislation, throughout the experiments.