Ecl chemiluminescent substrate

Oroxin B (OB, HY-N1435, purity: 99.71%, CAS number: 114482-86-9), Dimethyl sulfoxide (DMSO, HY-Y0320, purity: ≥99.0%, CAS number: 67-68-5), and autophagy inhibitor 3-methyladenine (3-MA, HY-19312, purity: 99.83%, CAS number: 5142-23-4) were acquired from MedChemExpress (Monmouth Junction, NJ, USA). Recombinant mouse IL-1β was purchased from R&D systems (Minneapolis, USA). Boster (Wuhan, Hubei, China) provided the RIPA lysis buffer, protease inhibitors, phosphatase inhibitors, BCA protein assay kit, protein loading buffer, rabbit/mouse secondary antibodies, and DAPI reagent. The ECL chemiluminescent substrate was obtained from Bio-Rad (Hercules, CA, USA). The biotechnology of Biosharp Life Sciences (Hefei, Anhui, China) provided the Trypsin (BL527A) and collagenase II (BS164). DMEM/F12 medium culture was got from Hyclone (Logan, UT, USA). Foetal bovine serum (FBS) was purchased from BioInd (Biological Industries, Israel). Omega Bio-tek (USA) provided the RNA extraction kit (R6834-01). Yeasen (Shanghai, China) provided the kits for complementary DNA (cDNA) synthesis (11141ES60) and quantitative real-time polymerase chain reaction (RT-qPCR) (11201es08).

To detect EBOVpp production, purified EBOVpp were processed using RIPA buffer (Sigma) supplemented with cOmplete Mini Protease Inhibitor Cocktail (ROCHE; Basel, Switzerland) and the protein concentrations were determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories; Hercules, CA, USA). Following which, samples were resolved by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto a nitrocellulose membrane by standard Western blotting techniques. The blots were then probed using the primary anti-EBOV GP (subtype Zaire, strain Mayinga 1976) rabbit polyclonal antibody (Sino Biological; Beijing, China) at 1:1000 dilution, and HIV-p17 monoclonal antibody (Santa Cruz Biotechnology; Dallas, TX, USA) at 1:200 dilution, followed by the respective horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam; Cambridge, United Kingdom) at 1:3000 dilution and detection using ECL chemiluminescent substrate (Bio-Rad Laboratories). Images were taken with a ChemiDoc-It^TS2 imager (UVP; Upland, CA, USA).

Equal amounts of protein from each sample were separated on SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 5% skim milk in TBST buffer (20 mM Tris, 137 mM NaCl, and 0.1% Tween 20, pH 7.4) for 30 min at RT and incubated with the appropriate primary antibody overnight at 4 °C. After several washes with TBST, the membranes were incubated with the corresponding IgG-HRP secondary antibodies at a dilution of 1:10,000 for 1 h at RT, washed, and visualized with the ECL chemiluminescent substrate (Bio-Rad, Hercules, CA). Unedited, expanded western blots provided in Supplementary Information. Several western blots were cut prior to antibody hybridization for reagent conservation.

Cellular lysates of unstimulated and BCG-stimulated DCs were prepared with the help of a M2 lysis buffer. Protein concentration in lysates was determined with Pierce BCA protein assay kit. 10–15 µg protein was loaded per well in 12% polyacrylamide gel and resolved at 30 mA. Subsequently, proteins were transferred onto 0.2 µm PVDF membrane at 120 mA for 2 h. Membranes were blocked overnight with 1% bovine serum albumin in Tris-buffered saline (TBS) at 4°C and thereafter, probed with anti-Bcl-2 (1:1000), anti- Bcl-x_L (1:2000) and anti-beta-actin (1:10,000) antibodies (Cell Signaling Technology, cat no. 2876, 2764 and 8457, respectively). After washing with TBST, membranes were incubated with secondary antibodies at 25°C for 2 h. After repeated washing, blots were developed using ECL chemiluminescent substrate according to instructions given by the manufacturer (Bio-Rad) and images were acquired using ChemiDoc Imaging System. Densitometry was performed using ImageJ software (NIH).

The midbrain and the striatum tissue lysates were prepared in RIPA buffer. Protein extraction from supernatants was performed using a ReadyPrepTM protein extraction kit (Catalogue No. #163–2086, Bio-Rad Inc.). Protein was determined in samples using the Bradford Protein Assay Kit (Catalogue No. #SK3041, Markham, Ontario, Canada). Equal protein concentrations from all samples were loaded with sample buffer 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris HCl. After boiling for 5 min, samples were loaded on polyacrylamide gel (SDS-PAGE) (Catalogue No. #161–0181, Bio-Rad Inc). A sandwich of PVD and gel was transferred with transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol), allowing transfer from the gel to the membrane using the BioRad Trans-Blot Turbo apparatus. After membrane blocking for 1 h, incubation with primary antibodies was performed at 4 °C. Afterwards, the solution was added to the HRP-conjugated secondary antibody (Novus Biologicals, USA). Then, the ECL chemiluminescent substrate (Catalogue No.# 170–5060, Bio-Rad, Inc.) was added. The chemiluminescence was captured, and image analysis was performed against β-actin using a ChemiDoc MP imager.