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).