Swift membrane stain kit

For assessment of newly synthesized proteins we used in vivo puromycin labelling46 (link)62 (link). Briefly, mice (14–15 weeks of age) were deprived of food for 6 h and refed for 2 h. Propofol (18 μl g⁻¹) was intraperitoneally injected in mice after 70 min of refeeding. After 10 min, mice were intraperitoneally injected with puromycin (0.04 μmol g⁻¹ body weight) and killed 35 min later. Excised muscles were processed for western blotting with anti-puromycin antibody (KeraFAST EQ-001, 1:1,000 dilution). Puromycin-labelled proteins were normalized to total proteins of the same blot stained with the Swift Membrane Stain kit (G-Biosciences).

For measurement of protein synthesis we used an in vivo SUnSET technique [24 (link),25 (link)]. Briefly, mice (13 weeks of age) were food deprived for eight hours. Propofol (18 μl/g) (Abbott Laboratories, North Chicago, United States) was administered via an intraperitoneal injection 15 minutes before the puromycin injection. The mice were then given an intraperitoneal injection of puromycin (0.04 μmol/g BW) and sacrificed 35 minutes later. At 10 minutes before sacrificing, insulin or saline (control) was administered via intraperitoneal injection. Muscles were isolated, homogenized, and prepared for western blotting with anti-puromycin antibody. For normalization to total protein, the same western blots were stained with Swift Membrane Stain™ kit (G-Biosciences, St. Louis, United States).