Enzyme-linked immunosorbent assay
The concentrations of mouse IL-1β, mouse TNF-a, mouse IL-6, human IL-1β, human TNF-a, and human IL-6 were measured using ELISA kits according to the manufacturer’s instruction.
ImmunofluorescenceImmunofluorescence analysis of NF-κB/p65 was conducted as previously described(Gao et al., 2016). Briefly, 2 × 105 RAW264.7 or THP-1 cells were seeded into a 35-mm glass-bottom SPL confocal dish overnight. The cells were pretreated with YDZG for 1 h, then stimulated with LPS (1 μg·mL-1) for another 2 h. Then, the primary antibody anti-NF-κB/p65 (1:100) and secondary antibody goat anti-rabbit Alexa Fluor 568 (1:200) were applied. Cells were fixed and stained with Hoechst 33342 (1 μM) for imaging. The immunofluorescence analysis of Caspase-1 p20 was conducted in a similar manner to NF-κB/p65, but the primary antibody anti-Caspase-1p20 (1:200) and secondary antibody goat anti-rabbit Alexa Fluor 488 (1:200) were applied. HEK293T cells transiently transfected with plasmids encoding GFP-NLRP3 were cultured for 48 h. Nuclei were stained with Hoechst 33342 (1 μM). Cells were examined using a confocal laser microscope (Leica, Wetzlar, Germany).
Immunoblotting and immunoprecipitationProtein samples were collected from treated cells, and protein concentrations were examined using a BCA protein kit (Waltham, MA, USA). Cytoplasmic and nuclear proteins were extracted according to the kit manufacturer’s instructions (Beyond time, Shanghai, China). For immunoprecipitation, the antigen samples were combined with specific antibody, then incubated overnight at 4 ºC with mixing. Protein A/G Magnetic Beads were washed and collected with a magnetic stand, then incubated with antigen sample/antibody mixture. After 1 h of incubation, beads were washed three times with IP buffer. Immunoprecipitates were eluted using SDS-PAGE reducing sample buffer. For immunoblot analysis, an equivalent amount of each protein sample was subjected to SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Membranes were then blocked with 5% non-fat milk dissolved in Tris-buffered saline Tween 20 buffer for 1 h, then probed with primary antibodies (1:1000) overnight at 4 °C. Corresponding secondary antibodies (1:5000) were then added for a further 1 h at 25 °C. Protein band signals were detected using SuperSignal West Femto maximum sensitivity substrate (Pierce Biotechnology) under visualization in a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).