Anti α synuclein antibody

Total proteins were extracted from the cell lysates using RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA, 1:50 dilution). Protein concentrations of the extracts were measured with a BCA assay kit. For Western blot, samples were mixed with sodium dodecyl sulfate (SDS) loading buffer and separated on 8%,10%, 12%, or 15% SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membranes were probed overnight at 4 °C with the following primary antibodies: anti-α-synuclein antibody (Abcam, Cambridge, UK, ab138501, 1:1000 dilution); anti- p62 antibody (Abcam, ab91526, 1:1000 dilution); anti-LC3B antibody (Novus, St. Charles, MO, USA, NB100-2220, 1:1000 dilution); anti-HDAC4 antibody (Proteintech, Wuhan, China, 17449-1-AP, 1:600 dilution); anti-β-actin antibody (Antgene, Wuhan, China, ANT009, 1:1000 dilution). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution) for 1 h at room temperature, western blots were revealed through chemiluminescence. The protein levels were quantified through densitometry using ImageJ v1.47 software.

Details are discussed in previously published literature [7 (link)]. Briefly, PC12 cells were plated on noncoated 12 mm coverslips and treated with MPP+ (0.5 mMol/L, 24 h exposure) and Paeoniflorin (50 uM, 24 h) and then fixed in ice-cold 4% paraformaldehyde for 15 min. The cells were then exposed to primary anti-LC3 antibody (1 : 250, Abcam, Cambridge, UK) or anti-α-synuclein antibody (1 : 250, Abcam, Cambridge, UK) for 1.5 h at 37°C. After appropriate secondary antibody (1 : 500, Cy3-labeled Goat Anti-Rabbit for anti-LC3, FITC-labeled Goat Anti-Mouse IgG for anti-α-synuclein, Beyotime Institute of Biotechnology, Jiangsu, China) treatment for 1 h, the labeled cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, 0.3 μg/mL) for 15 min and evaluated by a Laser Scanning Confocal Microscope (Leica TCS SP2 CLSM). Images were collected and processed using the imaging software provided by the Leica TCS system.