Capase 1

Fifty micrograms of extracted proteins were directly loaded into a sodium dodecyl sulfate (SDS) sample buffer for 10% SDS-polyacrylamide gel electrophoresis. After transferring the samples onto a nitrocellulose membrane (Trans-Blot Transfer Medium; Bio-Rad, Hercules, CA, USA), the membranes were blocked with 5% milk in a Tris buffer solution containing 0.1% Tween 20 for 1 h and then incubated with antibodies against Capase-1 (Santa Cruz Biotechnology, Dallas, TX, USA, EUA) or AIM2 (Cell signaling, Cat. 12498S, Danvers, MA, USA) overnight at 4 °C. Next, the cells were incubated with an IgG horseradish peroxidase (HRP)-conjugated secondary Ab (Cell Signaling, Cat.7076S, Danvers, MA, USA) for 1 h at room temperature. After the membranes were rinsed, the immunocomplexes were developed using an enhanced peroxidase/luminol chemiluminescence reaction (ECL Western blotting detection reagents, Pierce Biotechnology, Waltham, MA, USA) and exposed to an X-ray film with autoradiography (Carestream Health, Rochester, NY, USA). The bands were quantified densitometrically using the ImageTool 2.0 software (University of Texas, Austin, TX, USA), and the results were expressed as arbitrary units.

Tβ4 was purchased from Tocris Bioscience (Bristol, UK). Primary antibodies against LC3A/B, SQSTM1/p62, mammalian target of rapamycin (mTOR), phosphorylated (p)-mTOR, protein kinase B (AKT), p-PI3K, PI3K, p-AKT, p-JNK, JNK, IL-1β, TNF-ɑ, p-p38, and p38 were purchased from Cell Signaling Technology (Beverly, MA, USA). NLRP3, p-IL18, IL-18, p-NF-kB, NF-kB, and β-actin were obtained from Abcam (Cambridge, UK). Capase-1 was purchased from Santa Cruz (CA, USA). ASC was purchased from Invitrogen (Middlesex County, MA, USA). Secondary antibodies (i.e., anti-rabbit, anti-goat, or anti-mouse IgG antibody conjugated with horseradish peroxidase) were obtained from Millipore (Temecula, CA, USA). In addition, LPS and adenosine 5′-triphosphate (ATP) were purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade.