2.8. Western blot
After modeling, the total protein was extracted by Radio
immunoprecipitation assay (RIPA) buffer (Biosharp, BL504A, China), and
protein concentrations were measured with a Bicinchoninic Protein Assay
Kit (Biosharp, BL521A, China). The protein samples were analyzed in
10-15% SDS-PAGE with a voltage from 80-130 V, and then, transferred to
0.45μm polyvinylidene fluoride (PVDF) membranes (220 mA, 30-60 min). The
protein bands were blocked with TBS containing 0.05% Tween-20 and 5%
skim milk for 1-3 h at room temperature and incubated overnight at 4 ºC
with the following primary antibodies: GAPDH (CST, D16H11, Boston,
USA;1:3000), NLRP3 (Abcam, AB 109414, Shanghai, China; 1:500),
Pro-Caspase-1 (CST, D7F10, Boston, USA;1:1000), Cleaved Caspase-1(CST,
D57A2, Boston, USA;1:500). Next, wash off excess primary antibodies with
TBST. HRP-conjugated secondary antibody (Servicebio, GB23303, Wuhan,
China;1:3000) was incubated with horseradish peroxidase conjugate at
room temperature for 1h. After washing the membranes again with TBST,
the bands were analyzed with an ECL system (P10501; Applygen) and Image
J software (Bio‐Rad).