Western Blot Analysis of PKR Expression

Muscle and liver tissues were homogenised in lysis buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, 2% sodium dodecyl sulphate (SDS), protease and phosphatase inhibitor mixture (Roche, Basel, Switzerland) and centrifuged at 1000 × g for 10 min at 4 °C to discard cellular debris. Western blot analyses were performed as described.^{6 (link)} For PKR expression studies, gastrocnemius muscle from adult WT and transgenic mice were homogeneised in lysis buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X100, protease and phosphatase inhibitor mixture (Roche). A total of 1.5 mg of lysate was incubated overnight at 4 °C with 4 μg of antibody against total PKR. The complexes were pelleted with protein A–Sepharose (Invitrogen, Carlsbad, CA, USA), and then separated by SDS-PAGE with an antibody against PKR. Details on the antibodies are provided in Supplementary Table S3.