Las 4000 instrument

Cells were harvested in cold PBS and lysed with RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% NP40, 0.5% DOC, and 0.5% SDS) containing protein inhibitor cocktail (1:1000) on ice. They were then incubated at 4 °C for 30 min followed by centrifugation (16,000 × g, 4 °C, 10 min). The supernatant was collected and subjected to SDS-PAGE or native-PAGE (IRF-3 dimerization). Proteins were transferred to an Immobilon-P PVDF membrane (Millipore). The membrane was blocked in TBS-T containing 5% skim milk (blocking buffer) at RT for 30 min. Primary antibodies were diluted in blocking buffer at 1:1000 or 1:500 (cFLIP) and incubated at 4 °C overnight. Horseradish peroxidase (HRP)-conjugated secondary antibodies were diluted at 1:3000 in blocking buffer or TBS-T (cFLIP) and incubated at RT for 1 h. Protein signals were visualized using Chemi-Lumi One Super (Nacalai Tesque) and the LAS-4000 instrument (Fujifilm).
Antibodies were from: Cosmo Bio, anti-IRF-3 mouse mAb (CBX-CBX00167); Enzo Life Sciences, anti-cFLIP mouse mAb (#ALX-8040961-0100); Santa Cruz Biotechnology, anti-PKR mouse mAb (#sc-6282), anti-GAPDH mouse mAb (#sc-32233); Cell Signaling Technology, anti-TRIF rabbit mAb (#4596S), anti-PARP rabbit mAb (#9542S), anti-caspase 8 mouse mAb (#9745S), anti-caspase 9 rabbit mAb (#9502S), HRP-linked anti-mouse IgG (#7076S), and HRP-linked anti-rabbit IgG (#7074S).

Frozen WATs and hippocampi (n = 3–4 mice per group) were homogenized in T-PER lysis buffer (Thermo Fisher Scientific, Carlsbad, CA, USA) with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). After bicinchoninic acid assay (Thermo Fisher Scientific) for protein concentration, proteins were loaded and electroblotted. Blots were probed with primary antibodies (Supplementary Table S1). α-tubulin and β-actin were used as internal controls to normalize protein content in tissue samples. Protein bands were detected using enhanced chemiluminescence substrates (Pierce, Rockford, IL, USA), and chemiluminescence was analyzed using an LAS-4000 instrument (Fujifilm, Tokyo, Japan). The Multi-Gauge V 3.0 image analysis program (Fujifilm, Tokyo, Japan) was used for densitometry analysis.

Purified proteins were subjected to 15% SDS/PAGE and then transferred to polyvinylidene fluoride membranes (Bio-Rad), which were then blocked with 5% (w/v) skim milk in Tris-buffered saline with 0.1% Tween 20 detergent (TBST) (500 mM Tris, pH 8.5; 150 mM NaCl; 0.05% Tween 20) for 1 h and incubated with α-Syn primary antibody [sc-52979 (Santa Cruz Biotechnology) at a 1:750 dilution factor] for 12 h at 4 °C. The membrane was rinsed with TBST, incubated with anti-mouse IgG peroxidase secondary antibody (Sigma; 1:2500 dilution factor), washed again with TBST, and reacted with chemiluminescent substrate solution (ThermoFisher Scientific, SuperSignal West Pico Chemiluminescent Substrate) for 1 min. Resulting bands were detected using an LAS-4000 instrument (Fujifilm Life Science).