E ab 20059

Western blotting was performed as described previously (Ballesteros-Plaza et al., 2013 (link)) with some modifications: E. coli cells were sonicated. E. coli cell (1 × 10⁸ CFU) lysates were boiled in 5× loading buffer [0.25M Tris-HCL (pH 6.8); 10% (w/v) sodium dodecyl sulfate; 0.5% (w/v) bromophenol blue; 50% (v/v) glycerol; 5% (w/v) β-mercaptoethanol], and proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes using eBlot Protein Transfer Device (Genscript), according to manufacturer’s instructions. Membranes were probed with anti-E. coli AmpC Ab (mouse polyclonal antiserum; for details of preparation, see Supplementary Data) and anti-GAPDH Ab (1:1000 in PBS, rabbit polyclonal, #E-AB-20059, from Elabscience) followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse Antibody (1:5000 in PBS, #bsm-0294M-HRP, Bioss, China). Chemiluminescence was developed using an Immobilon Western chemiluminescent HRP substrate (Millipore) and documented using a Tanon 5200 milti (Biotanon). Densitometry was performed using ImageJ software (Ver 1.4.3.67) and was normalized to GapA (probed by anti-GAPDH Ab) (Wu et al., 2012 (link)).

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).