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).