Complete mini protease phosphatase inhibitor cocktail

Muscle samples from the in vivo study were harvested and digested in lysis buffer with complete mini protease/phosphatase inhibitor cocktail (Cell Signaling Technology, USA) according to instruction by the manufacturer. Harvested cells for in vitro studies were lysed using radioimmunoprecipitation assay buffer with complete mini protease inhibitor cocktail (Roche, USA) and soluble protein was collected by centrifugation at 18 407 rcf for 10 min at 4°C. Western blot analysis was performed according to previous protocol.24 Antibodies used were as follows: β‐catenin (1:3000; Cell Signaling Technology) and β‐actin (1:4000; Sigma, USA). Results were visualized on the GeneGnome XRQ (Syngene, Cambridge, UK) for the in vivo study or X‐ray film (Fujifilm, Japan) for the in vitro study.

TA muscle samples and C2C12 cells were homogenized and lysed in radioimmunoprecipitation assay (RIPA) buffer with complete mini protease/phosphatase inhibitor cocktail (5872S, Cell signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. Western blot analysis was performed according to our previous protocol [15 (link)]. Primary antibodies used were GAPDH (1:2000, MA5-15738, Invitrogen, Waltham, MA, USA), PI3 Kinase p85 (1:2000, 4292, Cell signaling Technology, Danvers, MA, USA), Akt (1:2000, 4691, Cell signaling Technology, Danvers, MA, USA), pAkt (1:2000, 4060, Cell signaling Technology, Danvers, MA, USA), mTOR (1:2000, 5043, Cell signaling Technology, Danvers, MA, USA), p70-S6K (1:2000, 34475, Cell signaling Technology, Danvers, MA, USA), EIF4EBP1 (1:2000, 9644, Cell signaling Technology, Danvers, MA, USA) and MyoD (1:2000, 13812, Cell signaling Technology, Danvers, MA, USA). Secondary antibodies used were anti-rabbit IgG, HRP-linked antibody (1:5000, Cell signaling Technology, Danvers, MA, USA) and goat anti-mouse IgG (H + L) antibody (1:5000, Invitrogen, Waltham, MA, USA). Relative protein contents were imaged by GeneGnome XRQ (Syngene, Cambridge, UK) and quantified by ImageJ.