Phospho protein kinase b akt ser473

Total protein of cells was extracted by lysis buffer and concentration determination was conducted using the DC protein assay method of Bradford (Bio-Rad, Hercules, CA). Twenty mg of protein was loaded and separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA). The antibodies used for the analysis are as follows, OPCML (Abcam, Cambridge, UK), cyclin-dependent kinase inhibitor 1B (p27), cleaved caspase 3, cleaved caspase 9, poly ADP-ribose polymerase (PARP), phospho-Protein Kinase B (AKT) (Ser473) and phosphor-glycogen synthesis kinase 3β (GSK3β) (Cell Signaling Technology, Inc., Danvers, MA), and GAPDH (Good Here Biotechnology, Hangzhou, P.R. China).

Approximately 30 to 40 mg of muscle tissue was minced in 10 μL/mg of homogenization buffer (50 mM Tris-HCL, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 50 mM sodium fluoride, and one protease inhibitor tablet per 10 mL of homogenization buffer, pH 7.5) on ice using small scissors and a Teflon pestle before being shaken for 10 minutes at 1500 rpm at room temperature and centrifuged at 11,000 rpm at 4°C for 5 minutes. The supernatant containing the sarcoplasmic proteins was transferred to a 2-mL eppendorf for western blotting according to our previous work (35 (link)). The remaining myofibrillar pellet was processed and analyzed for ¹³C₆ enrichment according to our previous work (35 (link)). Primary antibodies used for western blotting were: phospho p70S6K1 Thr389 (#9205), total p70S6K1 (#9202), phospho–eukaryotic initiation factor 4E binding protein (4E-BP1) Thr37/46 (#9459), total 4E-BP1 (#9452), phospho–eukaryotic elongation factor 2 (p-eEF2) Thr56 (#2331), total eEF2 (#2332), phospho–protein kinase B (Akt) Ser473 (#3787), and total Akt (#9272) from Cell Signaling Technology (New England Biolabs Ltd, Hitchin, United Kingdom).