2.3 Plethysmography, measurement of ATP concentration in cerebral spinal fluid (CSF), Western-blot and CD73 activity assay
All of the experimental protocols in this section have been described detailed in a previous study from this laboratory (8). Briefly, on the 29th or 30th day, rats were anesthetized with intraperitoneal injection of a mixture of anesthetics (urethane 0.84 g·kg-1, α-chloralose 42 mg·kg-1 and borax 42 mg·kg-1, dissolved in saline), and airway vagal activity was estimated by plethysmographic measurement of inspiratory resistance of the airway (Ri), expiratory resistance of the airway (Re) and dynamic compliance of the lungs (Cdyn) using a pulmonary function analyzing system (AniRes2005, Beijing Biolab Co. Ltd., Beijing, China). CSF was collected with a microsyringe punctured into the cisterna magna, and ATP concentration in CSF was measured with the luciferin-luciferase method using the Labsystems Luminoskan TL Plus luminometer (Thermo Scientific, Waltham, MA, USA) in combination with an ATP assay kit (S0026, Beyotime Biotechnology, Shanghai, China). After decapitation, brain tissue containing the NA and the middle lobe of the right lungs were isolated and put in 200 µL RIPA lysis buffer (P0013C, Beyotime Biotechnology) containing a protease inhibitor cocktail for general use (P1006, Beyotime Biotechnology), and homogenized. After centrifugation, the supernatant was collected and the concentration of total protein was measured using a microplate reader (Infinite M1000 Pro, Tecan, Switzerland) in combination with a BCA protein assay kit (CoWin Biosciences, Beijing, China). Samples with equal amount of protein were mounted on a running gel of 10% or 12% SDS-PAGE electrophoresis, and transferred onto a polyvinylidene fluoride membrane (Millipore, MA, USA). After incubation in a blocking buffer (TBST containing 5% w/v non-fat milk), the blots were incubated with a primary rabbit antibody against CD73 (ab175396; 1:2000; Abcam, UK), eosinophil cationic protein 1(ECP1 or EAR1)(bs-1754R; 1:2000; Bioss, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH,1:10000, 10494-1-AP, Proteintech Group, Inc., Chicago, USA), respectively, overnight at 4℃. This was followed by incubation with peroxidase-labeled goat anti-rabbit IgG secondary antibody (A0208; 1:2000; Beyotime Biotechnology) at room temperature for 2 h. Specific antibody-antigen complex was detected using an enhanced chemiluminescence Western-blot detection system (P10100, NCM Biotech, Suzhou, China) and imaged using the image analyzer (TanonImage, Tanon, Shanghai, China). General protein from the NA-containing brain tissue was isolated, and CD73 activity assay was performed using a microplate reader (Infinite M1000 Pro, Tecan) in combination with a CD73 activity assay kit (A041-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).