Chemiluminescent blocker

For Western blot analysis 50 μg of protein was loaded onto a 12% polyacrylamide gel Mini‐ PROTEAN TGXTM (BIO‐RAD, Milan, Italy) followed by electrotransfer to nitrocellulose membrane Trans‐Blot TurboTM (BIO‐RAD, Mylan, Italy) using Trans‐Blot SE Semi‐Dry Transfer Cell (BIO‐RAD). Subsequently, membrane was blocked in chemiluminescent blocker (Millipore, Darmstadt, Germany) for 1 h at room temperature. After blocking, the membrane was three times washed in phosphate‐buffered saline (PBS) for 5 min and incubated with primary antibodies against human MCT1 (ab90582, Abcam, Mylan, Italy), MCT4 (ab234728, Abcam), and β‐actin (ab181602, Abcam). Next day, after three washes in TBST, the membranes were incubated with antimouse (1:3000, Jackson, WestGrove, PA) and anti‐rabbit HRP‐conjugated (1:3000, Jackson, WestGrove, PA) secondary antibodies for 1 h at RT. Proteins bands were visualized with premixed ready‐to‐use chemiluminescent HRP detection reagent (Millipore) according to the manufacturer's instructions and captured using the C‐DiGit Blot Scanner (LI‐COR Biosciences, NE). The density of each band was quantified using ImageJ analysis software and normalized β‐actin levels measured in the same membranes.

For Western blot analysis 30 μg of protein was loaded onto a 12% polyacrylamide gel Mini- PROTEAN TGXTM (BIO-RAD, Milan, Italy) followed by electrotransfer to nitrocellulose membrane Trans-Blot TurboTM (BIO-RAD, Mylan, Italy) using Trans-Blot SE Semi-Dry Transfer Cell (BIO-RAD). Subsequently, membrane was blocked in chemiluminescent blocker (Millipore, Darmstadt, Germany) for 1 h at room temperature. After blocking, the membrane was three times washed in PBS for 5 min and incubated with primary antibodies against human CXCL12 (MA5-23759, Thermo Fisher Scientific), CX43 (#3512, Cell Signaling Technology, Danvers, MA, USA), and β-actin (ab181602, Abcam). Next day, after three washes in TBST, the membranes were incubated with antimouse (1:3000, Jackson, WestGrove, PA, USA) and anti-rabbit HRP-conjugated (1:3000, Jackson, WestGrove, PA, USA) secondary antibodies for 1 h at RT. Proteins bands were visualized with premixed ready-to-use chemiluminescent HRP detection reagent (Millipore) according to the manufacturer’s instructions and captured using the C-DiGit Blot Scanner(LI-COR Biosciences, Nebraska USA). The density of each band was quantified using ImageJ analysis software and normalized β-actin levels measured in the same membranes.

Snap-frozen muscle tissue was pulverized in a TissueLyser (Qiagen) for 2 × 20 s under liquid nitrogen using stainless steel beads. Tissue powder was homogenized in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate) at 1 ml/100 mg tissue containing 1:100 protease inhibitor cocktail (Calbiochem, USA) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, Germany). Determination of protein concentration was carried out using a BCA Assay Reagent Kit (UP95424 Uptima, France). Protein was denaturated by boiling, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to 2-μm nitrocellulose membranes (Bio-Rad, France) using a semi-dry Transblot Turbo system (Bio-Rad, France). After using a chemiluminescent blocker (Millipore, France), membranes were probed with primary antibodies against Glycogen Synthase Rabbit mAb (Cell Signaling; Cat#3886; 1:500), Phospho-Glycogen Synthase Ser641 (Cell Signaling; Cat#3891; 1:500), PGC-1α (Millipore; Cat#AB3242, 1:1,000), and total actin (Sigma-Aldrich; Cat#A2103, 1:10,000). Primary antibodies were detected with anti-rabbit HRP (P.A.R.I.S; Cat#BI2407, 1:5,000). The protein bands were detected by chemiluminescence using ECL Lumina Forte (Millipore) and a chemiluminescence detector (Bio-Rad, France).