Cias1 nalp3

Cell culture medium and human primary renal PTECs were provided by ScienCell (San Diego, CA, United States). Anti-SLC2A9 (URAT1) antibodies, TSG101, CD63, caspase-1, GSDMD, IL-1β, caspase-3, caspase-8, caspase-9, cytochrome c, Bcl-2, and Bax were purchased from ABclonal (Wuhan, China). Oxonic acid (OA) and UA were provided by Sigma (St. Louis, MO, United States). CIAS1/NALP3, GAPDH, and anti-GLUT9 were provided by Abcam (Cambridge, United Kingdom). In addition, the CCK-8 assay kit was provided by Jiwei Biological Technology (Shanghai, China).

To measure inflammasome protein expression, 2- and 4-h cultures of the different conditions (treated with P. gingivalis, P. gingivalis LPS, ATP 3 h + P. gingivalis or ATP 3 h + P. gingivalis LPS) were extracted in SDS sample buffer and separated by PAGE using gradient 5 to 12 % mini-gels, transferred to nitrocellulose membranes (Bio-Rad) and blocked overnight with 3 % BSA (Sigma) in 0.1 M Tris buffered salts solution pH 7.4 (TBS). Blotted antigens were incubated with rabbit polyclonal anti-human antibodies CIAS1/NALP3, TMS1/ASC, Caspase-1 (1 μg/ml, Abcam, Cambridge, UK), and β-actin (0.1 μg/ml, GenTex, Zeeland, MI, USA) as a loading control in 0.05 % Tween20/TBS for 4 h, washed three times and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit IgG, DAKO) diluted 1:1500 in Tween20/TBS for 2 h. Bound antibody was visualized with AP substrate (Bio-Rad) after development of reactivity for proteins from control antibody.