Cs 9662

For immunoblotting, snap frozen muscle samples were homogenized in 6 μL/mg muscle of ice cold lysis buffer with protease and phosphatase inhibitors and then centrifuged at 15,000xg for 40 min at 4°C. Supernatant was stored at -80°C until assayed for protein content using the bicinchoninic acid technique with BSA as a standard. Twenty-five μg of mixed muscle protein lysate were resolved on 4-12% SDS-PAGE gels and transferred to PVDF membranes. To assess inflammatory/proteolytic and anabolic signaling, antibodies for phosphorylated (Ser536) (CS-3033) and total NFκB p65 (CS-8242), TNF receptor 1 (TNFR1; CS-3736), caspase-3 (CS-9662), phosphorylated (Ser421/Thr424) (CS-9204) and total p70S6K (CS-2708), ribosomal protein S6 (CS-2217) and eIF2Bɛ (CS-3595) were purchased from Cell Signaling Technologies (Danvers, MA). Primary antibodies were diluted 1:1000 in PBST and 5% goat serum (monoclonal antibodies) or 2% milk/2% BSA (polyclonal antibodies). HRP-conjugated secondary antibody (Pierce Thermo Scientific, Rockford, IL) was used at 1:50,000 (w/v) followed by chemiluminescent detection in a BioRad (Hercules, CA) ChemiDoc imaging system with band densitometry performed using BioRad Quantity One software (software package 4.5.1) as detailed previously ^{34 (link)}.