Anti aif1

Protein samples were extracted from carotid arteries with a lysis buffer. The protein concentration was determined by the BCA method (Beyotime Institute of Biotechnology, Jiangsu, China). All samples were separated with different concentrations of SDS–PAGE according to different molecular weights, and transferred to an Immun-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA), membranes were blocked in 5% BSA (Solarbio, Beijing, China) in 1xTBST and were then incubated with anti-β-Actin (CTS, 8H10D10), anti-Ptprc (20103-1-AP; Proteintech, Rosemont, IL, USA), anti-Itgb2 (10554-1-AP; Proteintech, Rosemont, IL, USA), anti-Itgax (GB11059; Servicebio, Wuhan, China), anti-Ctss (Santa Cruz, sc-271619), anti-Ly86 (Santa Cruz, sc-390613), anti-CD44 (GB112054; Servicebio, Wuhan, China), anti-Aif1 (Santa Cruz, sc-32725) primary antibodies with working concentration about 1 μg/mL, and a horseradish peroxidase–conjugated secondary antibody, respectively. The bands were visualized using the Super Western Sensitivity Chemiluminescence Detection System (Thermo Fisher, Waltham, MA, USA). Autoradiographs were quantified by densitometry (NIH Image J).

For western blotting analysis, total proteins were collected from striatum, SN, and BV2 cells and stored at −80 °C. 40 ug of total protein lysate was loaded onto a 12% sodium-dodecyl-sulfate polyacrylamide gel in each lane, then transferred onto a polyvinylidene-difluoride membrane (Millipore, United Ststes). The primary antibodies were incubated overnight at 4 °C included anti-TH (1:500; Santa Cruz, sc-25269), anti-solute carrier family 6 member 3 (SLC6A3) (1:1,000; ABclonal, A152360), anti-dopamine receptor D2 (DRD2) (1:1,000; ABclonal, A12930), anti-AIF1 (1:1,000; Santa Cruz, sc-32725), anti-GFAP (1:1,000; ABclonal, A14673), anti-NLRP3 (1:200; AdipoGen, AG-20B-0014-C100), anti-PYCARD (1:1,000; Immunoway, T0365), anti-CASP1 (1:1,000; ABclonal, A0964), anti-IL1B (1:1,000; ABclonal, A12688), anti-MAP1LC3B (1:1,000; ABclonal, A11282), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738S) and anti-ACTB (1:3,000; ABclonal, AC026). HRP-conjugated anti-Rabbit antibody (1:5,000; ABclonal, AS014) or anti-Mouse antibody (1:5,000; ABclonal, AS003) was used as secondary antibody. ImageJ software was used to quantify the target bands and ACTB as an internal control.