Gapdh primary antibody

Western blotting was performed to validate the up-regulation of beta-actin abundance between ACL control and exercise groups using previously established methods 43 . In brief, 10 µg of ACL control and exercise samples were electrophoresed and separated on a pre-cast 12 well gel (Bio-Rad Criterion 10% TGX). Separated proteins were then transferred to a nitrocellulose membrane and blocked with 10 ml LICOR Odyssey ® blocking buffer (LI-COR, Cambridge, UK) for 1 hour at room temperature. Subsequently primary antibody (β-actin, Abcam, ab8227, Cambridge, UK) was added to the membrane at a 1:1000 dilution directly into the buffer and allowed to incubate overnight at 4ºC. The membrane was washed and incubated in a secondary goat anti-rabbit (LI-COR, IRDye® 680RD Goat anti-Rabbit IgG, Cambridge, UK) at 1:20,000 dilution was incubated for one hour at room temperature. The membrane was imaged by Odyssey ® LI-COR CLx imaging system at a wavelength of 700 nm. As a normalising control, GAPDH was also probed following the same steps as above using GAPDH primary antibody (Sigma, Poole, UK) at a 1:1000 dilution with goatanti rabbit secondary at 1:20,000 (LI-COR, IRDye® 800CW Goat anti-Rabbit IgG, Cambridge, UK). ImageJ software (http://rsbweb.nih.gov/ij/) was used to quantify bands using densitometry. Results were normalized to GAPDH loading control as reported previously 44 .

Muscle samples were weighed and homogenised in ice-cold homogenisation buffer (20 mM-HEPES, 1 mM-EDTA and 250 mMsucrose; pH 7•4) using a Polytron tissue homogeniser (Brinkmann Instruments). Protein contents in the homogenate were quantified by the Lowry protein assay, and an equal amount of protein was denatured and separated on 7•5 % SDSpolyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (New Life Science Product, Inc.). Non-specific binding sites on the membranes were blocked with TRIS buffer (10 mM-TRIS-HCl and 100 mM-NaCl, pH 7•5) containing 5 % non-fat dry milk at 4°C overnight. The blocked PVDF membranes were incubated with GLUT4 primary antibody (1:4000; Chemicon) and GAPDH primary antibody (1:5000; Sigma). Antigen-antibody complexes were visualised, detected and quantified using the ECL Western blot detection kit (Amersham Pharmacia Biotech), Luminescent Image Analyzer (Fujifilm) and Zero-Dscan densitometric analysis (Scanalytics, Inc.), respectively. Total expression of muscle GLUT4 protein content was normalised by the expression of total GAPDH level from the same gel.