Ab110411

Differentiated SGBS adipocytes and undifferentiated control cells were washed once with PBS, and then suspended in lysis buffer which consists of 50 nM Tris-HCl, 0,1% Triton X-100 (Sigma), 15 mM 2-mercaptoethanol (Sigma), 1 mM EDTA (Sigma) and protease inhibitor (Sigma). The lysates were suspended in 5x Laemmli loading buffer and boiled for 10 min at 100 °C. Equal amount of proteins were loaded onto a 12%-SDS-polyacrylamide gel, and transferred onto a PVDF Immobilon-P Transfer Membrane (Merck-Millipore, Germany). Then the membranes were blocked in 5% skimmed milk (Sigma) for 1 hour. For overnight, membranes were probed at 4 °C with primary antibodies: monoclonal anti-UCP1 (1:1000, R&D Systems, MAB6158), polyclonal anti-UCP1 (1:500, Sigma, U6382; 1:500, Thermo Scientific, PA1-24894), anti-OXPHOS (1:1000, Abcam, UK, ab110411), and anti-actin (1:10000, Sigma, A2066), in TBS-T containing 1% non-fat skimmed milk, followed by the incubation with horseradish-peroxidase-conjugated species corresponding secondary antibodies (Sigma) for 1 hour at room temperature. Immunoreactive proteins were visualized by Immobilion western chemiluminescence substrate (Merck-Millipore). ImageJ software was used to carry out the densitometry measurements.

For immunoblotting, cells were lysed in a buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X‐100, 10% glycerol, 10 mM EDTA, and the protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). After 20 min. rotation at 4°C, the lysates were centrifuged at 10,000 × g at 4°C for 10 min. Protein extracts (30–60 μg) were separated on 4–20% Tris‐Glycine Gel (Life Technologies) and electron‐transferred to polyvinylidene difluoride or nitrocellulose membranes according to standard procedures. After blocking free binding sites with 5% non‐fat dry milk reconstituted in Tris‐buffered saline with 0.2% Tween‐20, the membranes were probed with various antibodies: mouse anti‐LC3B (1/2000, Enzo, #ALX‐803‐081‐C100) (Enzo‐life Sciences, Lyon, France), rabbit anti‐P62 (1/1000, Enzo, #BMLPW9860‐0100) mouse anti‐ P84 (1/1000, Abcam, #ab487), mouse anti‐VDAC (1/2000, Abcam, #ab34726), mouse anti‐Human Oxphos (1/1000, Abcam, #ab110411) and mouse anti‐tubulin (1/2000, Sigma, #T9026). Anti‐mouse and anti‐rabbit peroxidase‐linked secondary antibodies (GE Healthcare, Life Sciences) were used at 1/10,000 and 1/20,000 respectively, and signals were detected using a chemiluminescence system (Super signal^® West Femto, Thermo Scientific, #34095) (Thermo Fisher Scientific). The images acquired were analysed quantitatively using Image Studio 2.1 software (Li‐Cor, Lincoln, NE, USA).