Confluent L6 myoblasts into myotubes were 5 days in medium. 2 FBS differentiated cultured L6 myotubes were kept in a medium containing 2 FBS until use. Re prims wildtype MEF 7 uses embryonic day 13.5 isolated as described above. SIN1 prim Ren MEF and corresponding wild type were embroidered by B Su available and was isolated as described above. Cell lysis and Western blot. Unless otherwise indicated, the cells were erismodegib serum starved overnight and minutes with inhibitors or DMSO 0.1 min for 30 minutes before stimulation incubated with 100 nM insulin for 10 min. All inhibitors were either synthesized as described above or were from Calbiochem. The cells were lysed in lysis buffer, and then ice-scraping short sonication. The lysates were transferred from the gel by centrifugation, SDS-PAGE, transferred to nitrocellulose St Rt clarified Rt and immunoblotting with Cell Signaling Technology antique Rpern. Unless otherwise indicated, the cells were lysed in 300 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 1 Triton X-100, 0.02 NaN3, 20 nM microcystin, phosphatases 1 and 2 inhibitor cocktails Sigma Roche protease cocktail and 2 mM PMSF. 6A and 7A and S2A, the cells were lysed in lysis buffer cap. Cape pull down assay. L6 myotubes from a well of a six-well plate were lysed in lysis buffer II ceiling 300 as described above. 50 ll lysis buffer cap II and 20 free washed before detergent beads were. To 150 ll cap STEM clarified Rte lysate and added 4 8C overnight with stirring The beads were washed twice with wash buffer cap 400′ll and twice with 500 ll PBS.
The beads were boiled in sample buffer and SDS-PAGE protein retained analyzed by Western blot. All organizations are old Cell Signaling Technology at Il. AntieIF4E antique box that was from BD Biosciences tests kinase. Phosphorylation of histone H1 by PKC ng in a buffer containing 200 ml of recombinant kinase, 25 mM HEPES pH 7.5, 10 mM MgCl 2, 5 mM tested glycerol phosphate, 0.05 mg ml phosphatidylserine, 0, 03 of Triton X-100, 0.5 mg ml BSA, 2.5 mM DTT, 100 lm CaCl2, 1 IM 10 LDCs and 15 ml ATP IN LCI 32PATP c. Inhibitors have been in a series PD0325901 of four dilutions tested 10-600 pM lM and four measurements gel St were taken at each concentration. The kinase reaction was. By spotting onto nitrocellulose, which was 5 times with 1 M NaCl is to finish a Ure phosphorus T remaining radioactivity t On the nitrocellulose film quantified by imaging plate and IC50 values were fitting the data to a sigmoid Dose-response curve is determined using Prism software. PDK1, mTORC1 and mTORC2 were analyzed as described above. In the cell of the West. L6 myotubes were cultured and differentiated in 96-well plates. At Wells enseite plate was not used for the experiment, but were kept filled with the media. After stimulation, the cells for 15 min at 4 in PBS with formaldehyde and Catt MGTT were fixed. The cells were Washe 
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