Mitoprofile total oxphos rodent wb antibody cocktail

Protein lysates were prepared from pulverized pgWAT samples, ARC-enriched microdissections or pituitaries in RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors. Cleared supernatants were resolved on pre-cast gradient 4%-12% SDS-PAGE gels (Bio-Rad), transferred onto PVDF membranes (Millipore), and probed with the following primary antibodies: Actin (1:1,000, Sigma-Aldrich), HSL, HSL-PSer660 and HSL-PSer563 (1:1,000, Cell Signaling Technology); MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (1:1,000, AbCam); OPA1 (1:1,000, BD Bioscience); Perilipin A (1:1,000, Cell Signaling Technology); alpha-tubulin (1:1,000; Sigma-Aldrich). Detection was performed by enhanced chemiluminescence (Pierce). Analysis of different phosphorylation sites was conducted by immunoblotting two replicate set of samples in parallel and confirming matched loading controls. Band intensities were quantified using the ImageJ software. Expression of phosphorylated proteins were normalized against the total protein homologue content, while non-phosphorylated proteins were normalized against loading control (Tubulin or Actin).

Proteins were extracted with RIPA buffer containing an inhibitor cocktail (Roche, Sussex, UK) and protein concentration was determined using a Bradford dye-based assay (Bio-Rad, Hemel Hempstead, UK). Total protein (30 μg) was subjected to SDS–PAGE followed by immunoblotting with appropriate antibodies at the recommended dilutions. The blots were then incubated with peroxidase-linked secondary antibodies followed by enhanced-chemiluminescent detection using the Super Signal Chemiluminescence Kit (Thermo scientific). The following antibodies were used: PGC-1α (rabbit, 1 : 1000, Abcam, Cambridge, UK), MEF2 (rabbit, 1 : 500, Santa Cruz, Middlesex, UK), Phospho-S6 Ribosomal Protein (Ser235/236) (rabbit, 1 : 100, Cell Signaling Technology, UK), S6 Ribosomal Protein (5G10) (Rabbit, 1 : 100, Cell Signaling Technology), TFAM (rabbit, 1 : 100, Sigma, UK), NRF1 (rabbit, 1 : 500, Life Span Bioscience, Nottingham, UK), Cleaved caspase-3 (rabbit, 1 :1 000, Cell Signaling), GAD67 (mouse, 1 : 1000, Millipore, Watford, UK), MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (mouse, 1 : 250, Abcam, UK), LC3B (rabbit, 1 : 1000, Sigma), β-Tubulin (rabbit, 1 : 1000, Santa Cruz), Synapsin (1 : 1000, Synaptic System, Goettingen, Germany), Actin (1 : 5000, Sigma), Cyclophillin D (1 : 1000, Abcam), GLS2 (1 : 100, Abcam), PFKp (1 : 500, Abcam) and GADPH (mouse, 1 : 10 000, Sigma).

Blue-Native PAGE was performed as described previously [38 (link),39 (link)]. Briefly, 200 μg of mitochondria were isolated and solubilized using solubilization buffer A (50 mM Sodium Chloride, 50 mM Imidazole, 2 mM 6-aminohexanoic acid, EDTA 1 mM, pH 7) with digitonin (6 mg digitonin/mg mitochondria). Mitochondrial complexes were subsequently separated using a Criterion™ TGX™ Precast 4–15% gel (Biorad) and Running Buffer 10x Tris/Gly (Biorad), and transferred to a PVDF membrane using the Trans Blot Turbo (Biorad). To detect OXPHOS (super)complexes, the membrane was incubated overnight with the following primary antibodies: COXIV (Cell Signaling, #4844), UQCRC2 (G-10) (Santa Cruz, sc-390378) and MitoProfile^® Total OXPHOS Rodent WB Antibody Cocktail (Abcam, ab110413). Membranes were thoroughly washed with TBS-Tween and incubated for 45min with HRP-conjugated secondary antibody. Pierce ECL Western Blotting Substrate (ThermoScientific) was used to develop the membranes. Images were digitally captured by LAS4000 (GE Healthcare) and optical density was analyzed with ImageJ software. Coomassie blue staining was used as loading control.