Plasma membrane fractionation of the skeletal muscle

Mice that had been denied access to food overnight received DS20060511 (10 mg kg⁻¹), insulin (5 U kg⁻¹), or saline (vehicle control) via the inferior vena cava under isoflurane anesthesia, and 10 min later, the triceps surae muscle was excised. The plasma membrane fraction of each skeletal muscle specimen was prepared as described previously^48,49. Briefly, the triceps surae muscle was homogenized in Buffer A (20 mM HEPES, 1 mM EDTA, 1 mM PMSF, and protease inhibitor) containing 250 mM sucrose on ice. The muscle homogenate was centrifuged at 2000 × g for 10 min at 4 °C to remove any unhomogenized muscle fibers. The supernatant was then centrifuged at 19,000 × g for 20 min at 4 °C. The pellet was resuspended in 3 mL Buffer A, layered on a 6 mL sucrose cushion (38% sucrose in Buffer A) and centrifuged at 100,000 × g for 60 min at 4 °C. The membrane fraction recovered on top of the sucrose cushion was resuspended in Buffer A and centrifuged at 40,000 × g for 20 min at 4 °C. The pellet was designated as the plasma membrane fraction and subjected to immunoblotting.