Ab238979

The RIPA lysis buffer (Beyotime, China) was used extract total proteins from clinical tissues and cell lines. The proteins were separated by 10% sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the targeted protein bands were transferred onto PVDF membrane (Sigma, USA). Subsequently, the PVDF membranes were probed with the primary antibodies including anti-β-actin (#ab8226, Abcam, USA), anti-SOD2 (#ab13534, Abcam, USA), anti-NLRP3 (#ab214185, Abcam, USA), anti-CyclinD1 (#ab16663, Abcam, USA), anti-Cyclin E2 (#ab32103, Abcam, USA), anti-CDK2 (#ab32147, Abcam, USA), anti-CDK4 (#ab108357, Abcam, USA), anti-CDK6 (#ab124821, Abcam, USA), anti-Caspase 3 (#ab13847, Abcam, USA), anti-Bax (#ab32503, Abcam, USA), anti-Bcl-2 (#ab185002, Abcam, USA), anti-Caspase 1 (#ab238979, Abcam, USA) and anti-IL-1β (#ab33774, Abcam, USA). After that, the membranes were incubated with anti-Rabbit IgG antibody at 4°C for 24h, and the enhanced chemiluminescent (ECL) system was employed to detect the protein bands. Finally, all the protein bands were quantified by Image J software. The primers sequences of the target genes were listed in Table 1.

Cells were firstly lysed in RIPA buffer (Sigma-Aldrich, USA) and protein concentrations of each homogenate were measured using a commercial BCA protein assay Kit (Thermo Fisher Scientific, USA). 30 μg of proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then blocked in 5% skimmed milk in TBST, and stained overnight at 4 °C with primary antibodies against Nrf2 (1:1000, ab137550, Abcam), HO-1 (1:2000, ab13243, Abcam), eNOS (1:5000, ab76198, Abcam), NQO1 (1:10,000, ab80588, Abcam), caspase-1 (1:1000, ab238979, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab180799, Abcam), HMGB1 (1:10,000, ab79823, Abcam), or β-actin (1:5000, ab115777, Abcam). Samples were then stained with goat anti-rabbit or goat anti-mouse IgG secondary antibody (1:10,000, ab205718/ab205719, Abcam) at room temperature for 2 h. Bands were developed using chemiluminescence substance (Thermo Fisher Scientific, USA). The proteins were quantified using Quantity One software (Bio-Rad, USA).