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Proteins were diluted in Laemmli sample buffer, separated in a 12% SDS–PAGE, immobilized on nitrocellulose membrane (Protran, Whatman, GE Healthcare, Versailles, France) and blotted with specific primary antibody at 1/1000 dilution: anti-PKC zeta (Santa Cruz sc-216, Clinisciences, Nanterre, France), anti-phospho-PKC zeta (Santa Cruz sc-12894R, Clinisciences, Nanterre, France), anti-Actin (Santa Cruz sc-1616, Clinisciences, Nanterre, France), anti-GFAP (Dako, Z 0334) and anti-Receptor Interacting Protein (RIP3 Sigma-Aldrich PRS2283)
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They will then employ a sterile needle to deliver the B12 solution into the muscle tissue