Ag 20b 0014

Cell lysate proteins (30 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were first blocked in 5% fat-free milk in PBST (Phosphate buffer saline, 0.1% Tween 20) and then probed with primary antibodies in 1% fat-free milk in PBST. The following antibodies were used with the indicated dilution: anti-CASP1 (1:1,000, Adipogen AG-20B-0042), anti-CD36 (1:1,000, R&D Systems AF2519), anti-FCGR2B (1:1,000, Cell Signaling 96397), anti-IL1B (1:1,000, GeneTex GTX 74034), anti-MRC1 (1:1,000, Abcam ab64693), anti-NLRP3 (1:3,000, Adipogene AG-20B-0014), anti-TLR2 (1:500, R&D Systems AF1530), and anti-TLR4 (1:1,000, Santa Cruz sc-293072). Membranes were washed in PBST and incubated with the appropriate HRP-conjugated secondary antibodies (1:5,000) in 1% fat-free milk in PBST. After five washes, immunoreactivity was revealed by incubating membranes with the HRP SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific), and the signal was detected by the Chemidoc XRS system (Bio-Rad). Protein amount loading control and normalization were achieved by probing the membranes with α-tubulin (TUBA1A) (1:4,000, Sigma-Merck T5168) or β-actin (ACTB) (1:10,000, Sigma-Merck A5441) antibodies.