Macrophage Protein Extraction and Western Blot

Protein extraction from macrophages was performed using TRIzol reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer’s instructions. Briefly, after phase separation using chloroform, 100% ethanol was added to the interphase/phenolchloroform layer to precipitate genomic DNA. Subsequently, the phenol-ethanol supernatant was mixed with isopropanol to isolate proteins. The Bradford method was used to determine protein concentrations in the cell lysate. Equal amounts of protein were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated overnight with antibodies against GSDMD (Abcam, ab209845), CASP11 (abcam, ab180673), CASP1 (AdipoGen, AG-20B-0042-C100), murine IL-1b (R&D Systems, AF-401-NA), LC3A/B (Cell Signaling Technology, 12741) and GAPDH (Cell Signaling Technology, 2118). Corresponding secondary antibodies conjugated with horseradish peroxidase in combination with enhanced chemiluminescence reagent (Amersham, RPN2209,) were used to visualize protein bands. Densitometry analyses were performed by normalizing target protein bands to their respective loading control (GAPDH) using ImageJ software as previously described^{17 (link),19 (link)}.