Supported nitrocellulose membrane

Protein samples were resolved by SDS/PAGE and transferred to supported nitrocellulose membranes (Bio-Rad) for 2 h at 4°C in transfer buffer (25 mM Tris pH 7.4, 193 mM glycine, 20% methanol) as described [29 (link)]. After blocking membranes for 30 minutes at room temperature with 10% nonfat dry milk in PBS-T (1 × PBS/0.1% Tween-20), membranes were washed and incubated with a 1:1000 dilution of primary antibodies for 2 h in 5% nonfat dry milk. After three times washing, blots were incubated with secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase (1:10,000 dilutions). Proteins were visualized with the enhanced chemiluminescence detection system, ECL+ (Amersham Pharmacia) and exposed to X-ray film.

Cells were either lysed directly on culture plates and scraped or trypsinised first before lysis using cell lysis buffer described elsewhere [62 (link)]. Equal amount of proteins from lysates were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transfered onto supported nitrocellulose membranes (BioRad, Hertfordshire, UK). Non-specific binding were eliminated by incubating membranes with 1% w/v milk in tris- buffered saline Tween-20 (TBST) overnight. Membranes were then probed with the following antibodies according to the manufacturer’s recommended dilutions; ACCA (Cell Signaling, Danvers, MA, USA), p-ACCA (S⁷⁹) (Cell Signaling, Danvers, MA, USA), FASN (BD Biosciences, Franklin Lakes, NJ, USA), IGF-IR (Cell Signaling, MA, USA), p-IGF-IR (Y^1135/1136) (Cell Signaling, Danvers, MA, USA), AKT (Cell Signaling, Danvers, MA, USA) and p-AKT (S⁴⁷³) (Cell Signaling, Danvers, MA, USA). Membranes were washed and incubated with anti-mouse IgG HRP or anti-rabbit IgG HRP secondary antibodies. Membranes were washed and the signals were detected using SuperSignal West Dura and Femto Chemiluminescent Substrates (Thermo Scientific, Rockford, IL, USA). The bands were analysed using Image J (National Institutes of Health, Bethesda, Maryland, USA).

BMCs were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA). Muscle samples were lysed in RIPA buffer with a tissue homogenizer. Total protein concentrations were determined by the BCA assay, and measurements were made with a Glomax microplate luminometer (Promega, USA). Samples were resolved on an 8% polyacrylamide gel and transferred to supported nitrocellulose membranes (Bio-Rad Laboratories). Next, the membranes were blocked in 5% milk dissolved in TBS-T (20 mM Tris, 150 mM NaCl and 0.1% Tween 20). Then, the membranes were incubated overnight at 4 °C with primary antibodies against the following targets: p-eNOS (Ser1177) (cod 9571, Cell Signaling, USA), eNOS (cod 9572, Cell Signaling, USA), ERRα (cod sc-787, Santa Cruz, USA), VEGF (cod sc-507, Santa Cruz, USA), and vinculin (Sigma-Aldrich, USA). Then, the membrane was washed extensively with TBS-T, probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako, USA) and developed with ECL (Amersham Biosciences, Italy). Each experiment was performed on at least three different mice for each experimental condition. Densitometric analyses were performed using NIH ImageJ software.

Clarified cell lysates and TCA precipitated media were resuspended in LDS sample buffer (Thermo Life Technologies), resolved on 4–12% NuPAGE gels (Thermo Life Technologies) and transferred to supported nitrocellulose membranes (BioRad). Membranes were probed with diluted antibodies raised against GFP (rabbit, Abcam ab290) or γ-tubulin (mouse, Sigma, T5326). Donkey anti-mouse and anti-rabbit antibodies coupled to IR-dye 680 or 800 (LI-COR) was used to detect primary antibodies. Blots were developed using an Odyssey CLx Licor Instrument. Original uncropped blots are provided as a supplemental data file.