Protein Extraction and Western Blot Analysis

Protein was extracted from 100 mg of white skeletal muscle in 1 mL of RIPA buffer supplemented with phosphatase (PMSF and NA₃VO₄) and protease inhibitors (P8340, Santa Cruz) using the Precellys^® Evolution homogenizer coupled to a Cryolys cooling system (Bertin Technologies, Montigny-le-Bretonneux, France).

Soluble protein concentration was determined by the Bradford method, using bovine serum albumin (BSA) (Sigma Aldrich, Tres Cantos, Spain) for the standard curve. 20 μg of the soluble protein fraction were added to a loading buffer (containing SDS and β-mercaptoethanol), heated at 95°C for 5 min and run in a 12% polyacrylamide gel. The proteins were then transferred overnight to Immobilon^®-FL PVDF 0.2-μm transfer membranes (Merck Millipore Ltd., Tullagreen, Cork, Ireland) that had been previously activated in methanol. Total transferred protein was determined by a 5-min incubation with REVERT^TM Total Protein Stain (LI-COR, Lincoln, Nebraska, United States). The signal was read at 700 nm using the Odyssey Fc Imaging System (LI-COR). After total protein quantification, the membranes were blocked with Odyssey Blocking Buffer (diluted 1:1 in TBS) (LI-COR) for 1 h at room temperature before being incubated overnight at 4°C and in agitation with the corresponding primary antibody diluted in blocking buffer + 0.05% Tween20. The primary antibodies used were purchased from ABCAM (Cambridge, United Kingdom), as follows: rabbit polyclonal anti-COX IV antibody (ab16056; 1:1000), rabbit polyclonal anti-CS antibody (ab96600; 1:2000), mouse monoclonal anti-mitofusin 1 + mitofusin 2 antibody [3C9] (ab57602; 1/1000) and rabbit polyclonal anti-UCP3 antibody (ab180643; 1/500 for WM and 1/1000 for RM). The cross-reactivity of these antibodies with gilthead seabream was confirmed by the molecular weight of the bands. In the case of COX4a and CS, the blots were performed from the same membranes which were split prior to the primary antibody incubation. After washing with TBS-T, the membranes were incubated with the corresponding secondary antibodies diluted in blocking buffer + 0.05% Tween20 at 1:10000 dilution: IRDye^® 800CW Goat anti-Rabbit (925-32211), Li-Cor, Lincoln, Nebraska, United States) and IRDye^® 800CW Goat anti-Mouse (925-32210, Li-Cor, Lincoln, Nebraska, United States). After incubation, the membranes were washed with TBS-T and the fluorescence of the immunoreactive bands was measured at 800 nm using the Odyssey Fc Imaging System (LI-COR). Stripping was performed using a commercial stripping buffer (NewBlot PVDF 5X Stripping Buffer) (LI-COR).