Ecl detection system

Total proteins were extracted from lysates of THP-1 cells treated with plasma from active AOSD patients or healthy controls. The samples were run on 10% SDS-PAGE and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti-α-tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight. After washing with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA). Immunoreactive bands were incubated with an ECL detection system (Advansta, Menlo Park, CA, USA) and visualized by radiographic film. The band intensity was quantitated by ImageJ software as described previously [22 (link)]. The protein levels of NLRP3, caspase-1, IL-1β, and IL-18 were normalized to α-tubulin.

The antibodies (anti-HSPA8, anti-AIF, anti-14.3.3 ζ/δ, anti-Calreticulin, anti-HMGB1, anti-HPRT1 and Anti-PP2A A subunit) used in this study were from Cell Signaling Technology and were used as recommended by the supplier. Cell lysates were prepared from all available cell lines. After PBS washing, the pellets were re-suspended in Protein Extraction Reagent Type 4, and following sonication in ice, the lysates were clarified by centrifugation. The protein contents were measured using the Bradford assay and 20 μg of each sample was used for SDS PAGE separation and subsequent transferred on Hybond-C membranes (GE Healthcare Life Sciences). Primary antibodies were detected by secondary HRP-conjugated antibodies using the ECL detection system (Advansta). Whole lane normalization was used for quantitative investigation, as previously described by Colella et al. [23 (link)] and Gürtler et al. [24 (link)]. Experiments were performed in quintuplicates and the statistical analysis was performed using the paired t-test.

Owing to the poor correlation between serum levels of HMGB1 measured by ELISA and Western blot in SLE patients [21 (link)], we also measured plasma levels of HMGB1 by Western blot in 6 pAPS patients and 6 HCs. First, 1ul of plasma sample was diluted with 24 ul RIPA buffer and heated at 95°C for 5 minutes in SDS-loading buffer. Proteins were separated by 12% SDS-PAGE in running buffer (25mM Tris, 192mM glycine, 0.1% SDS). The gel was run at 70V for 30 minutes then at 100V until the blue dye front reached the bottom. The gel was transferred to polyvinylidene difluoride membrane (PVDF) in transfer buffer (50 mM Tris, 384 mM glycine, 20% methanol) at 300mA 1hr with the Mini TBC Electrophoretic Transfer Cell (BIO-RAD., Hercules, CA, USA). The membranes were blocked with 5% skimmed milk in TBST (150 mM NaCl, 20 mM Tris–HCl [pH 7.4], 0.1% Tween-20) at room temperature for 1hr and then probed with anti-HMGB1 rabbit antibody (1:5000, Abcam, Cambridge, MA, USA) at 4°C overnight. The membranes were washed 3 times with TBST, followed by incubation with peroxidase-conjugated secondary goat anti-rabbit IgG antibody (1:10000, Millipore, Darmstadt, Germany) at room temperature for 1hr. The membranes were washed 3 times with TBST again. Blots were developed by ECL detection system (Advansta, Menlo Park, CA USA) and exposed to X-ray film.

Western blot experiments were performed to analyze the effect of HPE_DMSO on phosphorylation of Extra-regulated kinase 1/2 (ERK 1/2). Cells, untreated and treated for 5 min, 1 h, 4 h, and 24 h with HPE_DMSO, were washed with phosphate buffer saline (PBS) and lysated by protein extract kit (Active Motif, Carlsbad, CA, USA) in accordance with manufacturer’s instructions. Extracts were resolved on Mini-protean TGX precast gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to PDVF membranes (Bio-Rad Laboratories) and probed with specific antibodies in accordance with the manufacturers’ instructions. Rabbit antibodies anti-ERK 1/2 and anti-p-ERK 1/2 (MyBioSource, San Diego, CA, USA) were used at 1:1000; rabbit antibodies anti-c-Fos and anti-p-c-Fos (Cell Signaling Technology, Danvers, MA, USA) at 1:500 and mouse anti-actin (Merck Life Science) at 1:1000. The secondary antibodies HRP-conjugate, both anti-rabbit and anti-mouse (Immunological Sciences, Rome, Italy) were used at 1:5000. The blots were revealed by ECL detection system (Advansta, Menlo Park, CA, USA). Image acquisitions were performed by ChemiDoc Instrument (Bio-Rad Laboratories).

When pups reached their ages, they were sacrificed, and the olfactory were rapidly removed. The tissue samples were homogenized using an ultrasonic wave (10 s, 3 times) in RIPA lysis buffer, which contained a cocktail of proteinase and phosphatase inhibitors (Roche). After centrifugation at 12,000 rpm for 15 min at 4°C, the protein-containing supernatants were collected. The protein concentrations were determined with a BCA-based kit (Pierce). Lysate samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis; then, the proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes (Bio-Rad). After blocked with 5% defatted milk in Tween/Tris-buffered saline (TBST) for 1 h at room temperature, the membranes were incubated with the primary antibody at 4°C overnight. The following primary antibodies were used: mouse monoclonal GAD67 (Chemicon^® 1:5000), mouse monoclonal CB (Sigma-Aldrich 1:1000), goat polyclonal CR (Abcam 1:2000), rabbit polyclonal PV (Abcam 1:1000) and mouse polyclonal β-actin (Sigma-Aldrich 1:5000). After washing in TBST, the membranes were incubated in a secondary antibody for 2 h at room temperature. All of the blots were detected by an enhanced chemiluminescence (ECL) detection system (Advansta). The scanned images were quantified with ImageJ (version 1.47) Software.

Subconfluent monolayers of A549 cells in 24-well plates were virus-infected at a multiplicity of infection of one PFU/cell. At the indicated times, cells were lyzed in 100 µl of Laemmli buffer and an aliquot was used for electrophoretic separation and immunoblot detection using the NS1- and NP-specific polyclonal rabbit antisera (anti-NP PA5-32242, Thermo Fischer Scientific, Waltham, MA, USA) and the ECL-system detection (Advansta, San Jose, CA, USA).