γh2ax

Skin tissue was lysed in RIPA lysis buffer and protein was collected under centrifugation at 12,000× g rpm for 15 min at 4 °C. Protein concentrations in skin tissues were measured. The protein sample was diluted with loading buffer and boiled at 100 °C for 5 min. The protein samples were then separated by SDS-PAGE (concentration: 10%). They were then transferred to the PVDF membrane. The membrane was sealed with blocking buffer (5% milk) for 1 h, and incubated overnight with primary antibodies in a 4 °C refrigerator. The primary antibodies included NLRP3, caspase-1, IL-1β, γH2AX, Nrf2 (1:1000, Affinity Biosciences, Cincinnati, OH, USA), 3-NT (1:1000, Millipore, Boston, MA, USA), 4-HNE (1:1000, Alpha Diagnostic International, San Antonio, TX, USA), HO-1, CAT, and anti-β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was then rinsed with TBST (0.05% Tween 20) three times and incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. Finally, ECL kit was used to indirectly detect protein expression level. All experiments were repeated at least three times. Image J v1.8.0 software was used to conduct grayscale analysis of protein expression bands.

Cultured cells and tumor tissues were lysed in RIPA Lysis Buffer (Beyotime) supplemented with phenylmethanesulfonyl fluoride (PMSF, Beyotime) to extract total protein. Nuclear protein was extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer’s instructions. Protein samples from each group were loaded on a 10% or 14% SDS-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Thermofisher), followed by blocking with 5% bovine serum albumin (BSA; Biosharp, China) for 1 h. Immunodetection was conducted with antibodies against SH3RF2 (1:300, Santa cruz, USA, #sc-100976), cleaved poly ADP-ribose polymerase (PARP; 1:500, Affinity, China, #AF7023), cleaved caspase 3 (1:1000, Affinity, #AF7022), phosphorylated histone H2AX (γH2AX; 1:500, Affinity, #AF3187), RNA binding protein with multiple splicing (RBPMS; 1:1000, Proteintech, USA, #15187-1-AP), β-actin (1:20000, Proteintech, #66009-1-Ig), and Histone H3 (1:1000, Proteintech, #17168-1-AP). Secondary antibodies including horseradish peroxidase-conjugated goat antirabbit IgG and goat anti-mouse IgG (Proteintech, #SA00001-1 or #SA00001-2) were used at 1:10000 dilution. Protein bands were visualized using ECL reagent. Expression data were normalized to β-actin or Histone H3.