Protein Expression Analysis of Macrophages

Total cellular proteins were extracted from bone marrow-derived macrophages (BMDMs) or dorsal skin using RIPA buffer (ELPIS Biotech, Deajeon, Republic of Korea) with phosphatase and protease inhibitors, and 1 μg of protein was quantified using the Bradford assay [64 (link)]. The proteins were separated using 12% SDS-PAGE and moved to PVDF membranes. After blocking with 5% skimmed milk powder in 1X TBS containing 0.1% Tween-20 for 2 h, the PVDF membranes were incubated overnight at 4 °C with rabbit primary antibodies against NLRP3 (1:2000, AdipoGen Life Sciences, San Diego, CA, USA), mature IL-1β (IL-1β p17; 1:1000, Cell Signaling Technology), pro-IL-1β (IL-1β p31; 1:1000, Abcam, Cambridge, UK), cleaved caspase-1 (caspase-1 p20; 1:1000, AdipoGen Life Sciences), pro-caspase-1 (caspase-1 p48; 1:1000, Abcam), and β-actin (1:10,000, Bethyl Laboratories, Montgomery, TX, USA) [63 (link)]. The membranes were washed and then reacted with HRP-conjugated secondary antibodies at room temperature (20 ± 5 °C) for 2 h. The membranes were detected using enhanced chemiluminescence, and the target protein bands were observed manually by developing X-ray films or using the LAS-500 mini imager (General Electric, Boston, MA, USA). The expression levels of target proteins were analyzed using ImageJ (1.51j8).