Mouse anti β actin

Proteins were extracted from cells or tissues and added to the RIPA buffer. Cracking on ice for 30_60 minutes. The supernatant was collected by centrifugation at 4°C and 12000 rpm for 15 minutes. BCA protein assay kit (Boster Inc, China) was used for protein quantification. SDS-PAGE gel electrophoresis was performed. Then, the protein was transferred to the nitrocellulose membrane and 5% skimmed milk powder blocked the nonspecific antigen. The primary antibody was added and incubated at 4°C overnight. After washing the membrane, horseradish peroxidase-labeled IgG (secondary antibody) was added at 37 °C for 1h. Densitometric analysis was performed by Image Lab. In the assay, antibodies included rabbit anti-hnRNP E1 (Abcam, UK. ab74793, 1:1000), mouse anti-HPV16 E2 (Abcam, UK. ab17185, 1:1000), mouse anti-HPV16 E6 (Abcam, UK. Ab70, 1:1000), mouse anti-β-actin (Boster Inc, China, 1:300).

Tissues and cells were lysed in ice-cold RIPA lysis buffer (Solarbio, Beijing, China). Samples (30 μg per lane) were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA, USA). After the 1 h blocking with 5 % nonfat milk, membranes were probed with the following primary antibodies at 4° C overnight: rabbit anti-caspase 1 (1:1500; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-NLRP3 (1:1000; Biosynthesis Biotechnology, Beijing, China), anti-DDX3X (1:1000; Biosynthesis Biotechnology, Beijing, China), anti-IL-1β (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-IL-18, and mouse anti-β-actin (1:3000; BOSTER, Wuhan, China). After five washes with PBS-Tween 20, horseradish peroxidase (HRP)-conjugated goat antibodies (1:3000, Abcam, Cambridge, MA, USA) were added. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000; Biosynthesis Biotechnology, Beijing, China). These results were visualized and then quantified using Quantity One software (Bio-Rad, Hercules, CA, USA) by scanning the exposed X-ray films.

Western blotting was carried out as previously described [27 (link)]. The primary antibodies used in this study were as follows: rabbit anti-Ki-67, rabbit anti-TRAF6 (Millipore), rabbit anti-TAK1, rabbit anti-phospho-TAK1, rabbit anti-IκBα, mouse anti-phospho-IκBα (Cell Signaling Technology), and mouse anti-β-actin (Boster).