Extraction reagents

Total protein was extracted using RIPA lysis buffer, and nuclear protein was extracted using Extraction Reagents (Pierce Biotechnology, Inc., Rockford, IL. USA). Protein concentrations were measured using a BCA protein assay kit (Beyotime Biotechnology, China). Protein samples (50 µg) were separated on 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% low-fat milk, and incubated with primary antibodies Nrf2 (1:200, Cat# ab92946, Abcam, UK), HO-1 (1:500, Cat# ab13243, Abcam, UK), NLRP3 (1:500, Cat# ab214185, Abcam, UK), caspase-1 (1 : 200, Cat# sc-392,736, Santa Cruz, USA), and IL-1β (1 : 1000, Cat# ab9787, Abcam, UK). The membranes were incubated with HRP-linked secondary antibody. The β-actin (1:1000, Cat# ab8227, Abcam) was used as an internal control. The bands were visualized by ECL (Thermo, Waltham, MA, USA), and were analyzed using the ImageJ software.

1 × 10⁶ RAW264.7 cells/dish were cultured in a 6 cm dish and treated with CoQ₀ (2.5-10 μM) with or without LPS (1 μg/mL) and ATP (5 mM) for the designated period. Posttreatment, all the cells were detached from the culture dish and washed one time in cold PBS. Then, cytoplasmic, nuclear, and total extracts were prepared following the protocols as given by extraction reagents (Pierce Biotechnology, Rockford, IL, USA). Taking bovine serum albumin as standard, the amount of protein in every sample was calculated using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Using 8-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), an equal volume (50 μg) of denatured protein samples was first electrophoresed and later transferred to polyvinylidene fluoride (PVDF) and left overnight. On a subsequent day, blocking of the membranes was done for 30 min at room temperature by using 5% nonfat dry milk. Following blocking at first, using primary antibodies, the membranes were incubated for 2 h and later with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody for 2 h (Pierce Biotechnology, Rockford, IL, USA). For measuring band intensities, a densitometric graph was developed by using commercial software (AlphaEase, Genetic Technology Inc., Miami, FL, USA) representing control as 100%.