Ab102522

An assay kit (catalog no. ab102522; Abcam) was used to monitor XO activity according to the manufacturer’s instructions. Briefly, tissues were homogenized in 100 mM Tris-HCl buffer (pH 7.5) and centrifuged (10,000 × g for 15 min in 4°C) to get the supernatants. The working solutions were added to 50-μl volumes of supernatants and were incubated at 37°C for 30 min. The fluorescence measurement was performed in a microplate reader (Tecan) at 570 nm at intervals of 10 min for 1 h. For the standard, XO was used as supplied in the kit, and XO activity was determined by comparing the fluorescence of the supernatants with that of the standards. The specific activity was expressed as nanomoles per minute per milligram of protein.

PO (156124) and febuxostat (FEB) (SML1285) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The primary antibodies used in the present study were purchased from Bioworld Technology (Nanjing, China) and were as follows: rabbit anti-collagen 3A1, rabbit anti-α-SMA, rabbit anti-fibronectin, rabbit anti-vimentin, rabbit anti-E-cadherin, rabbit anti-NLRP3, rabbit anti-ASC, rabbit anti-caspase-1, rabbit anti-IL-1β, rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and rabbit anti-TGFβ1 (BS7028, BS70000, BS90514, BS1491, BS1098, BS90948, BS6205, BS5641, AP0063, BS72410,BS91338). The assay kits for UA (ab65344), creatinine (ab65340), xanthine oxidase (XOD) (ab102522), IL-1β (ab197742), and IL-18 (ab216165) were purchased from Abcam (Cambridge, UK). The PrimeScript™ RT Master mix was obtained from Takara (Takara, Japan) and the FastStart Universal SYBR Green master mix was obtained from Roche (Basel, Switzerland).