Ab128915

After the protein extraction and quantification using the radio immunoprecipitation assay (RIPA) buffer and BCA protein determination kit (Sigma), 40 µg proteins were separated by using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, we transferred the proteins onto the polyvinylidene fluoride membranes (Millipore) and Western Blocker™ solution (Sigma) was employed for preventing the non-specific combination, followed by the incubation of primary antibodies (overnight at 4 °C) and secondary antibody (at the room temperature for 1 h). Ultimately, the immune binding was discerned using the ECL Western HRP Substrate (Millipore), and analyzed with ImageLab software version 4.1 (Bio-Rad, Hercules, CA, USA). The antibodies were from Abcam (Cambridge, UK): anti-ki67 (ab16667, 1:1000), anti-E-cadherin (ab40772, 1:1000), anti-Fibronectin (ab32419, 1:1000), anti-matrix metalloproteinase 9 (anti-MMP9; ab219372, 1:1000), anti-PD-L1 (ab228415, 1:1000), internal control anti-GAPDH (ab128915, 1:3000) and anti-rabbit IgG/HRP-linked secondary antibody (ab205718, 1:5000).

The NLRP1 activator muramyl dipeptide (MDP) (Selleck, USA) was then added at 100 μmol for 24 hours to the oxidative stress model according to the preliminary experiment and the published studies [21 (link)–24 (link)]. The cell viability determination was the same as that in Section 2.1.
Total protein from HTR-8/SVneo cells was prepared with RIPA lysing buffer. The sample proteins (20 μg) of the different groups were separated using 10% SDS-PAGE and transferred onto PVDF membranes. These membranes were incubated with a primary antibody, followed by incubation of the anti-rabbit IgG secondary antibody. Protein expression was detected with an enhanced chemiluminescence detection kit. GAPDH served as an internal control. The antibodies included those for IL-1β (Abcam, ab216995), pro-IL-1β (Abcam, ab216995), pro-CASP1 (Abcam, ab207802), CASP1 (Abcam, ab207802), Beclin-1 (Abcam, ab207612), LC3 (Abcam, ab51520), p62 (Abcam, ab211324), ATG5 (Abcam, ab108327), ATG7 (Abcam, ab52472), NLRP3 (Abcam, ab263899), NLRP1 (BioLegend, 679802), and GAPDH (Abcam, ab128915). An ECL chemiluminescent reagent and imaging system (Bio-Rad, USA) were used to display protein bands, with the collected images analyzed using Bio-Rad software.

A549 or SPC‐A1 cells after transfection were lysed in order to extract protein. The concentrations of protein samples were evaluated with the bicinchoninic acid assay kit (Pierce). All extracted proteins were isolated by 10% SDS‐PAGE (Bio‐Rad Laboratories) and later transferred to polyvinylidene fluoride membranes (Millipore). After being treated with primary antibody against MYBL2 (0.8 μg/mL, AV100748, Sigma‐Aldrich) or GAPDH (1/20 000, ab128915, Abcam) and secondary antibodies, immunoreactive bands were exposed via ECL method.

The Western analysis assays for p53, Vinculin, and GAPDH detection were performed as previously described (18 (link)). Briefly, mixtures of 1 mg ml⁻¹ cell lysates and the fluorescent master mix were heated at 95 °C for 5 min. The samples and all other reagents were dispensed into the microplates and capillary electrophoresis Western analysis was carried out with the ProteinSimple WES instrument and analyzed using the inbuilt Compass software (ProteinSimple). DO-1 mouse anti-p53 antibody (1:400, Santa Cruz sc-126), mouse anti-vinculin antibody (1:600, R&D Systems MAB6896), and rabbit anti-GAPDH (1:800, Abcam ab128915) were used.

Total cellular protein was isolated with RIPA buffer containing protease and phosphatase inhibitors. After centrifugation for 5 min at 4°C, the supernatant was collected and subjected to the quantification of total protein by the Bicinchoninic Acid Assay (Beyotime Institute of Biotechnology, Inc., Shanghai, China). Equal amounts of protein were separated by SDS-PAGE in 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore Inc.). Prior to overnight incubation with primary antibodies, the membranes were blocked with 5% nonfat milk at room temperature for 2 h. The membranes were rinsed with Tris-buffered saline containing 0.1% of Tween 20 (TBST) thrice and next incubated at room temperature for 1 h with a horseradish peroxidase–conjugated secondary antibody (ab205718; 1:5000 dilution in TBST; Abcam, Cambridge, UK, USA). Finally, the protein signals were detected by means of the SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce; Thermo Fisher Scientific, Inc.). Antibodies against FOXA1 (ab170933; Abcam) and GAPDH (ab128915; Abcam) (primary antibodies) were acquired from Abcam and were applied at 1:1000 dilution in TBST. Quantity One software version 4.62 (Bio Rad Laboratories, Inc., Hercules, CA, USA) was applied to analyze the densitometry.