Western blot analysis
After treatments, the cells were lysed in RIPA buffer containing
protease and phosphatase inhibitors on ice. The concentration of protein
was tested using the bicinchoninic acid (BCA) protein assay. Protein
samples (30 μg) were separated by 6%, 10% or 12.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and were transferred to
0.22-μm PVDF membranes, followed by blocking for 2 h at room temperature
with 5% dried skimmed milk in Tris-buffered saline with 0.05% Tween
20. The membranes were incubated with primary antibodies at 4 ℃
overnight, including CD36 (1:1000,
proteintech, Cat#18836-1-AP, RRID:AB_10597244), TLR4 (1:1000, santa,
Cat#sc-10741, RRID:AB_2240715), IκBα (1:1000, CST, Cat#4812), PCNA
(1:10000, proteintech, Cat#60097-1-lg, RRID:AB_2236728), NLRP3
(1:3000, CST, Cat#15101S, RRID:AB_2722591), cleaved-IL1β (1:1000, CST,
Cat#83186S, RRID:AB_2800010), cleaved-caspase-1 (1:1000,CST,
Cat#4199T RRID:AB_1903916), GAPDH (1:1000, ZSGB-BIO, Cat#ta-08,
RRID:AB_2747414). Subsequently, the membrane was incubated with
Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:8000)
for 2 h at room temperature. Immunoreactivity were visualized by
chemiluminescence method using ChemiDocTM MP Imaging System (Tanon,
China). The protein bands were quantified using a Bio-Rad Chemi EQ
densitometer and Bio-Rad Quantity One software(RRID:SCR_014280) and
normalized to GAPDH or PCNA.