P erk

Using Radioimmunoprecipitation assay buffer (RIPA) lysate, mouse tissues were lysed for 30 min on ice, and protein supernatants were collected by centrifuging at 4 °C and 12,000 rpm for 15 min. The concentration of protein was determined using the BCA assay. The proteins were denatured by adding a loading buffer and heating the mixture for 10 min. The total amount of protein was limited to about 40 μg. After 30 min of electrophoresis at 80 V, the voltage was increased to 120 V for an additional 90 min, and then electroconversion was performed. BSA blocked the PVDF membrane for 2 hours at room temperature. The required antibodies (ERK, proteintech, Cat No. 11257-1-AP, 1:1000; p-ERK, proteintech, Cat No. 28733-1-AP, 1:2000; JNK, proteintech, Cat No. 24164-1-AP, 1:1000, p-JNK, proteintech, Cat No. 80024-1-RR, 1:1000; P38, proteintech, Cat No. 14064-1-AP, 1:1000; p-P38, proteintech, Cat No. 28796-1-AP, 1:1000 and GAPDH, Elabscience, Cat No. E-AB-20059, 1:5000) were diluted in the primary antibody’s dilution solution and incubated overnight at 4°C.After three TBST treatments, the secondary antibody was added and incubated for 2 h at room temperature. The ECL luminescence developer was configured according to specifications, exposed using a chemiluminescence instrument, and the results were analyzed.

Immunofluorescence staining was performed as previously described [22 (link)]. Briefly, the cells and tissue sections were washed with PBS-0.25% Tween-20 (PBS-T) three times, blocked with 5% goat serum, and incubated with the primary antibodies rabbit anti-NLRP3 (1:200, CST, USA), caspase-1 (1:200, Abcam, USA), IL-1β (1:200, CST, USA), and PERK (1:200, Proteintech, China) overnight at 4 °C. Then, the cells and tissue sections were incubated with the fluorescent secondary antibody (goat anti-rat antibody or goat anti-rabbit antibody, Proteintech, China, diluted 1:100) for 1 h. Finally, the cells and tissue sections were counterstained with 4′-6-diamidino-2-phenylindole (DAPI) and observed with a fluorescence microscope. (Beyotime, China).
Pyroptosis was detected by propidium iodide (PI) staining following the manufacturer’s recommendations [23 (link)]. The quantification of fluorescence intensity was performed using ImageJ software.

SDS-Page electrophoresis and transfer of proteins to nitrocellulose membranes were performed according to standard procedures. Ponceau S (0.1%) staining of membranes verified transfer of proteins and equal loading. Membranes were incubated with antibodies to CD133 (Miltenyi Biotec, Auburn, CA, USA), cleaved and intact PARP, BAD, p-BAD, BCL-XL, MCL-1 (BioLegend, San Diego, CA, USA) cleaved active caspase-3, cleaved active caspase-9, BCL-2, active BAX (Novus Biologicals, Centennial, CO), AKT and p-AKT (Santa Cruz Biotech, Dallas, TX, USA), ERK, p-ERK, or to β-Actin (ProteinTech, Rosemont, IL, USA) as a loading control. Immunoblots were sequentially reprobed with other antibodies after stripping them of antibodies. Immune complexes were detected by incubation with horseradish peroxidase-conjugated antibodies to mouse or rabbit IgG (1:3000), followed by enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA) and imaging in a GE Healthcare Amersham Imager 600.