Bally 1

Isolated 1–3 × 10⁶ PBMCs and 0.5–1 × 10⁶ human vascular endothelial cells were pelleted and treated with homemade lysis buffer [48 (link)]. This is succeeded by measuring the protein concentration of the samples at 562 nm with a spectrometer (Tecan, Megelan, using Pierce^TM BCA Protein Assay Kit (Cat#23225, Thermo Fischer Scientific), where 20 µg of sample protein was subjected to SDS page electrophoresis and transferred to the PVDF membrane according to the standard laboratory procedure. We employed human primary antibodies for phospho-STAT-3 (Tyr 705) (D3A7), STAT-3 (D3Z2G), phospho-P65 (93H1), P65 (D14E12) (1:1000 or 1:2000), and cleaved caspase-1 (p-20) (Bally-1, Adipogen Life Sciences, San Diego, CA, USA) to detect the respective proteins. We used α-Tubulin (11H10) (Cell Signaling, Danvers, MA, USA) and/or β-actin (2D4H5) (Proteintech, Rosemont, IL, USA) as the loading protein and anti-rabbit IgG, HRP-linked antibody (Cat# 7074, Cell Signaling), and AP-conjugated anti-mouse IgG (S3721, Promega, Madison, WI, USA,) were used as secondary antibodies. As substrate, we used Pierce^TM ECL Western, Super Signal^TM West Femto, or BCIP/NBT (Promega). The subsequent development of bands was detected using an INTAS ECL ChemoStar Imager (INTAS science Imaging) and further densitometries were quantified using LabImage ID software.

PBMCs, monocytes or macrophages were lysed in (5×) Laemmli SDS buffer supplemented with 1% β-mercaptoethanol, 0.01% bromophenol blue and protease inhibitor cocktail (Roche). Equal amounts of protein were analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and incubated with primary and secondary antibodies following the manufacturers’ instructions. Primary antibodies against ASC (1:2000, AL177), caspase-1 (1:1000, Bally-1) and NLRP3 (1:1000, Cryo-2) were from Adipogen. Anti-IL-1β (1:500, AF201) antibody was from R&D. Anti-GAPDH and anti-Actin antibodies (1:5000) were from Cell Signaling. Secondary HRP-linked antibodies against rabbit and mouse IgG (1:2000) were from Cell Signaling and against goat IgG (1:2000) was from R&D. Immunoblots were revealed using the enhanced chemiluminescence reagent (Thermo Scientific) and visualized using a BIORAD camera (Universal Hood II). GAPDH or Actin was used as loading controls.