Rabbit anti il 1β

Whole‐cell lysates were generated using Laemmli sample buffer (Bio‐Rad) and diluted in SDS‐PAGE sample buffer. Cell lysates were separated in 10% or 15% SDS‐PAGE and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk and probed with the following antibodies: rabbit anti‐lamin‐B1 (1:5,000; Abcam), mouse anti‐lamin‐A/C (1:5,000; Active Motif), rabbit anti‐TNF‐α (1:1,000; Cell Signaling), rabbit anti‐IL‐1β (1:1,000; Cell Signaling), rabbit anti‐IL‐6 (1:1,000; Novus biological), rabbit anti‐IL‐1α (1:2000, Santa Cruz), and mouse anti‐β‐actin (1:4,000; Sigma, AC‐15). Antibodies were detected with HRP‐conjugated anti‐mouse (1:10,000) or anti‐rabbit (1:10,000) antibodies and West Pico Substrate (Thermo Scientific).

After indicated treatment, cells were harvested and washed twice with PBS. Then they were lysed in RIPA buffer (Solarbio, Beijing, China) containing complete protease and phosphatase inhibitor (Solarbio, Beijing, China). The level of protein concentrations in cells was measured with a bicinchoninic acid protein assay kit (Solarbio, Beijing, China). The proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes by a wet electrophoretic transfer method. The membrane was blocked with TBST containing 5% non-fat milk for 2 h at room temperature followed by incubation overnight at 4 °C with primary antibodies. After overnight incubation, the blots were washed three times with TBST and incubated with secondary antibodies for 1 h at room temperature. Finally, the membranes were scanned using a Bio-Rad Gel imaging system (Bio-Rad, Berkeley, CA, USA) after visualization treatment with the ECL reagent. The primary antibodies, rabbit anti-PBK, rabbit anti-KIAA0101, rabbit anti-TOP2A, rabbit anti-IL-1β, and rabbit anti-β-actin, were purchased from Cell Signaling Technology (Boston, MA, USA). The rabbit anti-ASPM and rabbit anti-MCM10 were obtained from Proteintech (Rosemont, Chicago, IL, USA). Quantitative assessment of the and intensity was performed using Image Lab statistical software.

Nigericin and LPS were purchased from InvivoGen Biotech Co., Ltd. (San Diego, CA, USA). Mouse anti-FLAG, mouse anti-myc, mouse anti-β-actin, mouse anti-HA, rabbit anti-IL-1β, rabbit anti-NLRP3, rabbit anti-Casp-1, and rabbit anti-ASC monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated anti-rabbit antibody or anti-mouse antibody were purchased from ZSGB-BIO, Lnc. The translation inhibitor cycloheximide (CHX) was purchased from APEXBIO (Houston, TX, USA). Lipofectamine 2000 was purchased from invitrogen (Waltham, MA, USA).