Nox4 14347 1 ap

Punicalagin (purity = 98%, CAS: 65995-63-3) was purchased from Herbpurify Co., Ltd. (Chengdu, China). A urea assay kit, a creatinine assay kit (sarcosine oxidase) and a urine microalbumin assay kit were purchased from the Nanjing Jiancheng Bioengineering Institute (China). A mitochondrial membrane potential assay kit with JC-1 and a tissue mitochondria isolation kit were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies targeting the following proteins were used in this study: NLRP3 (A12694, Abclonal), Trx (A01219-1, BOSTER), TXNIP (sc-166234, Santa Cruz; A9342, Abclonal), IL-1β (A1112, Abclonal), caspase-1 (BM4291, BOSTER), GSDMD (A10164, Abclonal), β-actin (60008-1-Ig, Proteintech), NOX4 (14347-1-AP, Proteintech).

Cancer cells treated with MIAM were lysed in buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 0.1% triton X-100, 1.5 mM MgCl₂, 1 mM EDTA, 2 mM sodium orthovanadate, 4 mM sodium pyrophosphate, 100 mM NaF, and protease inhibitor mixture (Roche) on ice. Equal amounts of proteins were separated by SDS-PAGE electrophoresis and then electrotransferred onto PVDF membrane, blocked with 5% nonfat dry milk for 1 h at room temperature. The levels of proteins were determined by incubation with primary antibodies with appropriate dilution overnight. The primary antibodies included those specific to Bax (sc-493), Bcl-2 (sc-492), p53 (sc-126), phospho-Rb (Ser 795) (p-Rb, sc-21875), p21^Waf1/Cip1 (sc-397), p22^phox (sc-20781), BIM (sc-11425), β-actin (sc-47778, Santa Cruz), NOX4 (14347-1-AP, ProteinTech), and SIRT3 (#2627, Cell Signaling). The bound antibodies were visualized by using an enhanced chemiluminescence reagent (Millipore) and quantified by densitometry using ChemiDoc XRS+ image analyzer (Bio-Rad).