Muscle Cell Fractionation and Analysis

Cell fractionation and analysis from gastrocnemius muscle were performed as described previously [62 (link)]. Briefly, 100 mg of gastrocnemius muscle was resuspended in 0.5 mL of ice-cold buffer B (150.0 mM NaCl, 10.0 mM TRIS HCl pH 7.4, 1.0 mM EDTA, supplemented with protease inhibitors), passed through a 21-gauge needle 20 times. A 50 µL quantity of the resuspended sample was stored by transferring it into a new 1.5 mL microcentrifuge tube and used as total homogenate. The remaining fraction was centrifuged at 400 rpm for 5 min at 4 °C. The resulting supernatant was centrifuged at 14,000 rpm for 10 min at 4 °C in an Eppendorf centrifuge to pellet the Heavy Membranes (HM). The supernatant was centrifuged at 45,000 rpm for 30 min at 4 °C in a Beckman Coulter TL-100 centrifuge (Brea, CA, USA) to pellet the Light Membranes (LM) and isolate the Cytosolic fraction. The collected fractions were resolved on linear 10% polyacrylamide gel and analyzed for the expression of anti-GLUT-4 primary antibody (anti-Glucose Transporter GLUT-4 [1F8], ab35826, Abcam), anti-CNX (anti-Calnexin, ADI-SPA-860-F, Enzo Life Sciences, Pero (MI) Italy) and anti-GAPDH antibody (Elabscience, cod. E-AB-20059). The intensities of the corresponding bands were determined and quantified with ImageJ 1.52k Wayne Rasband, NIH (Bethesda, MD, USA) (http://imagej.nih.gov/ij).