Mini protean 3 dodeca cell

Western blot was performed as described previously [22 (link)]. The brain samples were homogenized in lysis buffer containing 150 mM NaCl, 50 mM Tris, 1% Triton X-100, and protease inhibitors: 2 mM PMSF and 2 μg/mL leupeptin, pepstatin and aprotinin. Protein samples (50 μg) were separated via SDS electrophoresis in Mini-Protean 3 Dodeca Cell (Bio-Rad Laboratories, USA) in 12% polyacrylamide gel. The resolved proteins were transferred to 0.45-µm nitrocellulose membrane by Trans-Blot system (Bio-Rad Laboratories, USA). The proteins were stained with primary antibodies: rabbit monoclonal antibodies Iba-1 (1:500, EPR16589, ab178847, Abcam, Cambridge, MA), cleaved caspase-3 (1:500, #9664, Cell Signaling, USA), and rabbit polyclonal antibodies β-actin (1:20000, I-19, sc-1616, Santa Cruz Biotechnology, USA). Secondary anti-rabbit IgG (Bio-Rad Laboratories, USA) were used at 1:1000 dilutions for Iba-1, cleaved caspase-3 staining, and 1:10000 dilution for β-actin staining. The blots were developed with a SuperSignalTM West Femto Maximum Sensitivity Substrate chemiluminescence kit (Thermo Fisher Scientific, USA) and quantified after scanning with a ChemidocTM Touch Imaging System (Bio-Rad Laboratories, USA) using the Scion Image 4.0.3.2 program (Scion Corporation, USA). The levels of Iba-1 and cleaved caspase-3 proteins were expressed in relative units to β-actin in the same sample.

Cobrotoxin was a gift from the Department of Pharmacy at the Suzhou University Medical College (Suzhou, Jiangsu, China), while the 3-methyl adenine (3-MA), dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 was purchased from Beyotime Institute of Biotechnology (Haimen, China) and an automatic enzyme standard instrument (Benchmark) was purchased from Bio-Rad (Hercules, CA, USA). A Heracell 150 carbon dioxide incubation box was purchased from Thermo Fisher Scientific (Rockford, IL, USA). SDS-PAGE apparatus (Mini-PROTEAN Tetra Electrophoresis System and Mini-PROTEAN 3 Dodeca Cell, Bio-Rad) and a FEI TECNAI 10 transmission electron microscope (TEM; Philips, Amsterdam, Holland) were also utilized.

Lung fragments (0.05 g) were homogenized in ice-cold RIPA lysis buffer (Beyotime, China) and protein concentrations determined using the BCA protein Assay Kit (Beyotime, China) with the absorbance read on a Bio-Rad 680 microplate reader (Bio-Rad, USA). Proteins were separated using the 10 % SDS-PAGE method (Mini-protean 3 Dodeca cell, Bio-Rad, Hercules, CA). The protein was then transferred onto a polyvinylidenedifluoride membrane (Bio-Rad, Hercules, CA). A pre-stained marker was utilized to determine the separation of proteins. The membrane was probed with anti-Foxp3 or anti-RORγt monoclonal antibodies, followed by alkaline phosphatase-conjugated secondary antibodies (BD, USA). Protein was visualized using the brightening agent, ECLTM western blotting detection (GE healthcare, No. RPN2106), and protein band intensity was quantified using the Gel-Pro imaging software (Media Cybernetics, Silver Spring, MD).