Nucleospin rna protein purification kit

Total RNA was extracted from the myotubes using the NucleoSpin RNA/protein purification kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. The RNA was reverse transcribed using the Verso cDNA Synthesis Kit (Fisher Scientific, Illkirch, France). Quantitative PCR was performed in triplicate wells with gene-specific primers using the LightCycler 480 system (Roche, Basel, Switzerland) and SYBR Green 1 Master Mix (Roche) as follows: 40 cycles of amplification with 10 s at 95 °C, 20 s at 60 °C and 20 s at 72 °C. The list of the primers used in this manuscript is presented in Table S1. Ct values of the target gene were normalized to the Ct values of the housekeeping gene RPLP0. The expression level of each transcript was determined using the 2^−ΔΔCt method.

Proteins were extracted from the myotubes using the NucleoSpin RNA/protein purification kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. Proteins were separated by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore, Bedford, MA, USA). A protein extract was loaded on each gel as a calibrator but this calibrator lane was deleted for better visualization of the presented data in Figure 1 and Figures 3 and 6. Proteins of interest were revealed by specific antibodies: anti-α-tubulin 1/20,000 (Sigma-Aldrich); anti-MHC1 1/2000 (Sigma-Aldrich); anti-phospho-AKT 1/2000 (Cell Signaling Technology, Danvers, MA, USA); anti-AKT 1/2000 (Cell Signaling Technology), followed by a secondary antibody 1/30,000 (Eurobio Scientific, Les Ulis, France). Lipid peroxidation was detected by anti-4-hydroxynonenal antibody 1/400 (Millipore, Bedford, MA, USA). The membranes were analyzed on an Odyssey imaging system (LI-COR Biotechnology, Lincoln, NE) and the band densities were quantified using ImageJ software (National Institutes of Health).