*4.9. Western Blot Analysis*

Cells were seeded into 6-well plates (2 × 10<sup>6</sup> cells/well) and incubated in a 37 ◦C and 5% CO2 incubator for 12 h. Cells were then treated with di fferent concentrations of juglone for 2 h, followed by treatment with LPS (1 μg/mL) for 6 h and ATP (5 mM) for 1 h. Cells were then washed with cold PBS and lysed with RIPA bu ffer (Thermo Scientific, Waltham, MA, USA). Protein concentration was quantified using a BCA protein assay kit (Sigma). Proteins (40 μg) were separated by SDS-PAGE (10% and 15% gels) and transferred onto polyvinylidene difluoride membranes using a Mini Tran-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories). Membranes were blocked with 5% skimmed milk at room temperature for 2 h, and washed three times with 1× TBST bu ffer (20 mM Tris-HCl, 150 mM NaCl, and 5% Tween 20, pH 7.6). Membranes were incubated with the primary antibodies (1:1000 dilution) at 4 ◦C for at least 10 h. After three washes with 1 × TBST bu ffer, membranes were incubated with mouse or rabbit IgG-horseradish peroxidase conjugated secondary antibodies at room temperature for 2 h. Westsave ECL detection reagen<sup>t</sup> and a chemiluminescent Western blot imaging instrument (Labgear Australia, Milton, Australia) were used to visualize the signal intensities. NLRP3 (NACHT, LRR and PYD domains-containing protein 3; #13158), and caspase-1 antibody (#24232) were purchased from Cell Signaling (Danvers, MA, USA). IL-1β (sc-7884) and β-Actin (sc-130656) antibody were bought from Santa Cruz Biotechnology (Dallas, TX, USA). IL-18 antibody (A16737) was purchased from ABclonal (Woburn, MA, USA). Mouse anti-rabbit secondary antibody (sc 2357)/or anti-mouse IgG HRP secondary antibody (sc516102) were bought from bought from Santa Cruz Biotechnology (Dallas, TX, USA). All antibodies were used at a dilution of 1:1000.
