Il 1β p17

PBMCs were lysed with 200 µL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, USA) containing protease inhibitor (Roche, Germany). Then, 50 µg of total protein was loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After sealing with 5% skimmed milk at 37 °C for 120 min, the PVDF membranes were incubated at 4 °C overnight with the primary antibodies against capase-1 p10 (Abcam, 1:1,000), IL-1β p17 (R&D Systems, Minneapolis, MN, USA; 1:1,000), NLRP3 (Cell Signaling, 1:1,000), caspase-1 (Cell Signaling, 1:1,000), ASC (Cell Signaling, 1:1,000), p-p65 (Cell Signaling, 1:1,000), and p-IκB α (Cell Signaling, 1:1,000) or Lamin-B (Cell Signaling, 1:1,000). Following that, the membranes were incubated with goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 60 min. An automatic digital gel image analysis system Bio-Rad CFX-96 (Bio-Rad, CA, USA) was employed to determine and analyze the band densities.