Second antibodies

RIPA buffer was applied to extract total protein from either macrophages or foot pad tissues. Cytoplasmic and Mitochondrial Proteins were extracted using Cytoplasmic and Mitochondrial Protein Extraction Kit (Sango Biotech, Shanghai, China) following the manufacturer's instruction. Cell culture supernatants were collected and concentrated (×10) using Amicon Ultra Centrifugal Filters (MilliporeSigma). Proteins were suspended in Laemmli Buffer 1×, boiled at 95°C for 6 minutes, and resolved by SDS-PAGE. Then, proteins were transferred to PVDF membrane. Western blot was performed using the following antibodies: anti-IL-1β, anti-Phospho-NF-κB p65(Ser536), and anti-Phospho-NF-κB p105/50 (Ser933) were purchased from Cell Signaling Technology (CST, USA), and anti-COX-2, anti-iNOS, anti-MPO, anti-IκBα,, anti-MyD88, anti-Caspase-1, anti-TLR4, anti-NF-κB p65, anti-NF-κB p105/50, and anti-NLRP3 were obtained from HuaBio (Hangzhou, China). The membrane was incubated with the primary antibodies at 4°C overnight. After washing the membrane, the membrane was incubated with second antibodies (1 : 7500 dilution, Boster, Wuhan, China) for 1 h at room temperature. Finally, the membrane was exposed to the gel imaging system with a chemiluminescence kit. The band intensity was quantified using Image J software.

After different treatments, primary neurons were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for another 20 min, and then blocked with 5% bovine serum albumin in PBS for 1 h at room temperature. Subsequently, primary neurons were incubated with primary antibodies at 37 °C for 2 h: anti-phospho-synuclein alpha (Ser129) (cat. no. AF3285, 1:500, Affinity Biosciences), anti-VMAT2 (cat. no. PA5-112713, 1:200, Thermo Fisher Scientific), anti-VAMP2 (cat. no. DF6381,1:200, Affinity Biosciences), and anti-α-syn (cat. no. OM239190, 1:200, Omnimabs). Then, primary neurons were incubated with second antibodies from Boster Biological Technology Co., Ltd. for 1 h at room temperature. The nuclei were stained with DAPI. The fluorescence images were obtained by using a laser scanning confocal microscope (Olympus FV1200, Japan) and the Pearson’s correlation coefficient was calculated by using Fiji software (version 1.54 f).

Alpha‐modified minimal essential medium (α‐MEM) and foetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Scoresby). Corilagin (Figure 1A) with a purity greater than 98% was purchased from Tongtian Biotechnology Co. and dissolved in different concentrations of 0‐2 µmol/L with dimethyl sulphoxide (DMSO), respectively. Primary antibodies against ERK, JNK, p38, p65, IkBα, p‐ERK, p‐PI3K, TRAF6, p‐TAK1, p‐JNK, p‐p38, p‐p65, p‐IkBα, GAPDH, NFATc1/NFAT2, c‐Fos and c‐Src were purchased from Cell Signaling Technology, while second antibodies were purchased from Boster Biological Technology Co.. Recombinant RANKL and recombinant M‐CSF were obtained from Novoprotein Scientific Inc (Shanghai, China). Tartrate‐resistant acid phosphatase (TRAP) staining kit and all other reagents were purchased from Sigma‐Aldrich, unless stated otherwise.