Anti pro caspase 1

Commercial PM_2.5 (Diesel particulate matter; NIST^® SRM^® 1650b, Sigma-Aldrich, St. Louis, MO, USA) and tetrahydrochloride (NSC-23766; RAC1 inhibitor; N5412, Sigma-Aldrich) were purchased. The reagent 8-OHdG was donated by Myung-Hee Chung (Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Republic of Korea). CellRox^® oxidative stress reagents (Sigma-Aldrich) were used in the intracellular oxidative stress detection experiment. We also purchased anti-NOX1 (PA5-39281, Invitrogen, Waltham, MA, USA), anti-NOX2 (ab129068, Abcam, Waltham, MA, USA), anti-NOX3 (ab81864, Abcam), anti-NOX4 (ab109225, Abcam), anti-RAC1 (05-389, Millipore, MA, USA), anti-p22^phox (#27967S, Cell Signaling Technology, Danvers, MA, USA), anti-NLRP3 (#15101, Cell Signaling Technology), anti-pro-caspase-1 (#3866, Cell Signaling Technology), anti-cleaved-caspase-1 (#4199T, Cell Signaling Technology), anti-precursor-IL-1β (#12703, Cell Signaling Technology), anti-mature-IL-1β (#83186T, Cell Signaling Technology), and anti-ASC (A96472, Antibodies.com, St Louis, MO, USA) antibodies. β-Actin (SC-47778, Santa Cruz, TX, USA) was used as an internal control for the Western blot analysis. The secondary antibodies HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were obtained from Cell Signaling Technology. Human IL-1β, IL-6, and IL-18 ELISA kits were purchased from R&D (Santa Clara, CA, USA).

Cells derived from 10 NORM and 10 CKD-HD were lysed in RIPA buffer (1 mM phenylmethylsulphonylfluoride, 5 mM EDTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 8 μg/ml leupeptin, 1.5% NonidetP-40, 20 mM Tris–HCl, pH 7.4). Aliquots containing 45 μg of proteins from each lysate were subjected to SDS–PAGE on a Criterion Tris-HCl 4–20% precast gels and then transferred onto PVDF membrane (Millipore). Membranes were incubated with primary antibodies as follows: anti-cleaved-Caspase-1 (Cell Signaling), anti-proCaspase-1 (Cell Signaling), anti-cleaved-IL1β (Cell Signaling), anti-pro-IL-1β (Santa Cruz), anti-IL-18 (Santa Cruz), and anti-actin (Santa Cruz). Blots were subsequently incubated with secondary antibodies HRP-labelled (Santa Cruz Biotechnology Santa Cruz, CA). Proteins were detected by Chemiluminescence (Amersham, GE Healthcare). Images were acquired using a scanner EPSON Perfection 2580 Photo (EPSON, Long Beach, CA, USA) and quantified by Image J 1.34 Software (http://rsb.info.nih.gov/ij/). The intensity of bands of interest was normalized to the signal intensity of the corresponding procaspase-1, pro-IL-1β, or pro-IL-18 band present on the same membrane. Actin was used as loading control.