Lysis and extraction buffer

RIPA (Radio Immunoprecipitation Assay) Lysis and Extraction Buffer (Thermo Scientific ™) was added to frozen liver tissue samples, and they were grinded in a homogenizer. The obtained tissue homogenate was centrifuged at 15,000 rpm (10 min, 4°C) and the supernatant was left. Then the Pierce ™ BCA (bicinchoninic acid) Protein Assay Kit (Thermo Scientific ™) was used to determine protein concentration. Equal amounts of proteins were separated by 10–12% SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skim milk for 3 h on a shaker at room temperature and then incubated with the primary antibodies (diluted with 5% skim milk) overnight at 4°C, and the concentrations of the primary antibodies are as follows: the concentration of primary antibody used for GAPDH, p-AKT, AKT, Nrf2, HO-1, NLRP3, p-PI3K, PI3K, and Caspase-1 was 0.5 μg/ml, and the concentration of primary antibody used for PPARγ, and Caspase-3 was 1 μg/ml. The membranes were washed via Tris-Buffered Saline Tween-20 (TBST) three times for 10 min each time and then incubated with the secondary antibodies (diluted with TBST) on the second day. Finally, the membranes were visualized with the chemiluminescent HRP substrate after washed again by TBST and analyzed via Image J gel analysis software.

Skin tissues were harvested on days 14 and 21 after irradiation and cut into small pieces. The frozen tissue samples were resuspended in radioimmunoprecipitation assay (RIPA) Lysis and Extraction Buffer (#89900; Thermo Fisher Scientific, Waltham, MA, USA) and lysed using TissueLyser II (QIAGEN, Hilden, Germany). The lysates were centrifuged at 13,500× g for 20 min at 4 °C, and the supernatants were collected. The protein concentration was determined using the Bio-Rad DC Protein Assay Kit II (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins were separated via SDS-PAGE and transferred to a Protran^TM nitrocellulose membrane (GE healthcare, Piscataway, NJ, USA). After blocking with 10% skimmed milk for 70 min at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. The bands of interest were visualized using an enhanced chemiluminescence detection kit (GE healthcare) according to the manufacturer’s instructions. Protein bands were quantified using ImageJ (National Institutes of Health, Bethesda, MA, USA).