Bicinchoninic acid protein assay

Total protein was extracted by lysing cells in radio-immunoprecipitation assay (RIPA) lysis buffer (GenDEPOT, Barker, TX, USA) containing a protease inhibitor cocktail (GenDEPOT) for 30 min on ice. The protein concentration was determined using a bicinchoninic acid protein assay (Sigma-Aldrich). Equal amounts of protein were loaded onto a 12% (v/v) sodium dodecyl sulfate-polyacrylamide gel for electrophoresis. The separated proteins were blotted onto a polyvinylidene fluoride membrane (Amersham Biosciences, Piscataway, NJ, USA). Membranes were first blocked with 3% BSA in Tris-buffered saline with 0.1% Tween-20 at RT for 1 h and then incubated overnight at 4 °C with primary polyclonal antibodies (1:1000 dilution; Table 2). The blots were then incubated with peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (7076 and 7074, respectively, Cell Signaling Technology, Danvers, MA, USA) diluted at 1:10,000 for 1 h at RT. The target proteins were visualized using an enhanced chemiluminescence kit (Amersham Biosciences), imaged using an Amersham Imager 680 (Amersham Biosciences) and quantified with ImageJ software. The density of each band was normalized to that of β-actin.

Animals were sacrificed by decapitation and SNc area was rapidly removed and frozen on dry ice, and stored at −80 °C. Protein lysate was obtained by re-suspending SNc in ice-cold lysis buffer (CelLytic, Sigma, USA) containing diluted phosphatase (1:10, Roche, Monza, Italy) and protease inhibitors (1:25, Roche, Italy). After centrifugation, the supernatant was collected and protein concentration was measured using a Bicinchoninic Acid Protein Assay (Sigma, USA). Protein lysates were run on 10% gels, transferred onto nitrocellulose membranes (Biorad, USA) and western blot was performed. Membranes were blocked (Odyssey blocking buffer, LiCor, USA) and incubated overnight with the following primary antibodies: anti-GFAP (1:2000, Sigma, USA, G3893), anti-TRPV1 (1:500, Santa Cruz, USA); anti-CNTF (1:500, Millipore, Germany, MAB338). As secondary antibodies, anti-mouse 1:10000 secondary IgG HRP-Conjugated were used. Image analysis of western blots was performed using Azure 600 azure biosystems and software (LiCor, Biosciences, Rockville, MD, USA) and signal was normalized with the corresponding GFAP signal.

The activity of ACE2 was determined in abdominal aorta lysates using a fluorometric assay kit (Angiotensin II Converting Enzyme Activity Assay Kit, BioVision, Inc., Milpitas, CA, USA). Frozen aortae were homogenized using a glass Teflon homogenizer in lysis buffer in accordance with the manufacturer’s instructions, and lysates obtained by centrifugation at 16,000× g/10 min/4 °C were used to measure the protein concentration (bicinchoninic acid protein assay; Sigma-Aldrich, St. Louis, MO, USA) and ACE2 activity. The assay procedure utilizes the ability of active ACE2 to cleave a synthetic MCA-based peptide substrate to release free MCA (7-methoxycoumarin-4-acetate), quantified using a fluorescence microplate reader (Synergy™ H4 Hybrid Reader; BioTek, Winooski, VT, USA) and enzyme kinetic measurements over 120 min at room temperature with data collection in 5-minute intervals at wavelengths Ex/Em = 320/420 nm. Enzyme kinetics were measured in the absence (negative control) or presence of an ACE2-specific inhibitor to distinguish ACE2 activity from other proteolytic activities.

Activity of glutamyl aminopeptidase was determined in the membrane fraction. Skeletal muscle and epididymal adipose tissue were homogenized using a glass Teflon homogenizer in lysis buffer (250 mM saccharose, 10 mM Tris, pH 7.4). The homogenates were centrifuged at 1,000 × g/10 min/4°C. The supernatants were collected and centrifuged at 16,000 × g/15 min/4°C to separate the membrane fraction. Protein concentration was measured by the Bicinchoninic Acid Protein Assay (Sigma-Aldrich, St. Louis, MO, United States). Prepared samples were mixed with substrate solution containing 100mM H-Glu-β-naphthylamide (Bachem, Bubendorf, Switzerland), 10 mg/100 ml bovine serum albumin, 10 mg/100 ml dithiothreitol, 50 mM CaCl2 in 50 mM Tris pH 7.4. The 96-well plate was placed in a Synergy H4 Hybrid Reader (BioTek, Winooski, VT, United States) fluorimeter and the enzyme kinetics was measured during 60 min at 37°C with data collection in 5-min intervals as the amount of β-naphthylamide released from the substrate due to the enzyme activity of AP-A at wavelengths 340 nm (excitation) and 410 nm (emission).
Superoxide dismutase activity was measured in skeletal muscle tissue samples by colorimetric SOD Activity Assay Kit (Abcam, Cambridge, United Kingdom). Tissue homogenization and assay procedure were performed in accordance with the manufacturer’ protocol.