Sc 7884

Fetal bovine serum (FBS) and modified Eagle’s medium (MEM) were purchased from Gibco (Grand Island, N.Y, USA), and phosphate-buffered saline (PBS), Western Breeze Chromogenic Kit anti-mouse, anti-rabbit, and anti-goat, and LysoTracker Green DND-26 were bought from Invitrogen (Carlsbad, CA, USA). Kit lactic dehydrogenase-based, sulfanilamide, H₃PO₄, and N-1-naphthylethylenediamine dihydrochloride were obtained from Sigma-Aldrich, St. Louis, MO, USA. Antibodies for IL-1β (H153): sc-7884, IL-2 (C2-1-hIL-2): sc-32295, IL-6 (E-4): sc-28343, IL-8 (807): sc-52870, IL-18 (H-173): sc-7954, tumor necrosis factor-alpha (TNF-α) (N-19): sc-1350, cyclooxygenase-2 (COX-2) (M-19): sc-1747, nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) (H-300): sc-13032, matrix metalloproteinase-2 (MMP-2) (H-76): sc-10736, matrix metalloproteinase-9 (MMP-9) (M-17): sc-6841, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 (A): sc-109 and β-actin (C4): sc-47778 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Invitrogen (Carlsbad, CA, USA). All of the other chemicals used were of analytical grade and were purchased from Sigma (St. Louis, MO, USA).

Tissue extracts (120 μg) were resolved by 10% reducing SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes [37 (link)]. The membranes were blocked for 2 h at room temperature in 3% bovine serum albumin solution in 20 mM Tris–HCl, pH 7.4 containing 150 mM NaCl and 0.02% Tween 20 (TBST) followed by overnight incubation at 4 °C with 1:500 polyclonal anti-MMP9 (sc-6841, Santa Cruz Biotechnology), MMP3 (sc-6839, Santa Cruz Biotechnology), TNF-α (sc-1351, Santa Cruz Biotechnology), IL-1β (sc-7884, Santa Cruz Biotechnology), IL-17 (sc-374218, Santa Cruz Biotechnology), TGF-β (sc-7892, Santa Cruz Biotechnology,) and β-actin (4967S, cell signalling technology, MA, USA) antibodies. The membranes were washed four times with TBST and then incubated with their respective alkaline phosphatase-conjugated secondary antibody (Santa Cruz Biotechnology) (1:2000) for 1.5 h. The bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solution (Sigma).

Cell lysates from sorted macrophages were prepared for Western blot. Samples with equal amounts of protein (10 µg) were resolved on 15% acrylamide denaturing gels (SDS-PAGE). Proteins were transferred to nitrocellulose membranes (Trans-Blot Turbo Mini Nitrocellulose Transfer Packs, Bio-Rad, Billerica, MA, USA) using the Bio-Rad Trans-Blot Turbo Transfer System for 7 min. The membrane was blocked in 1× Tris buffered saline and 0.1% Tween 20 (TBS-T) containing 5% low-fat milk for 1 h and probed overnight at 4 °C with primary antibodies diluted in blocking buffer. Primary antibodies used were: β-actin 1:500 (MAB1501, Merck Millipore, Burlington, MA, USA), IL1β 1:250 (sc-7884, Santa-Cruz Biotechnology, Santa Cruz, CA, USA) to detect pro-IL1β and IL1β, and caspase-1 1:500 (Santa-Cruz Biotechnology, sc-56036) to detect pro-caspase-1 and caspase-1, followed by anti-mouse 1:5000 (Abcam, ab97023) and anti-rabbit 1:5000 (Abcam 97051) HRP-conjugated secondary antibodies. The immunoreaction was detected with SuperSignal™ West Pico Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA, USA) and visualized using BioRad ChemiDoc XRS system.

WB was performed according to our previous study method [17 (link), 35 (link)]. We used the following primary antibodies to perform the WB analyses: rabbit monoclonal anti-NF-κB p65 (D14E12) XP® antibody (1:1000, #8242, Cell Signaling Technology); rabbit polyclonal anti-Nrf2 (L593) antibody (1:500, BS1258, Bioworld); mouse monoclonal anti-Cryopyrin (NLRP3) (6F12) antibody (1:1000, sc-134306, Santa Cruz Biotechnology); rabbit polyclonal anti-PYCARD (ASC) antibody (1:500, A1170, Abclonal); goat polyclonal anti-caspase-1 p20 (M-19) antibody(1:1000, sc-1218, Santa Cruz Biotechnology); rabbit polyclonal anti-IL-1β (H-153) antibody (1: 1000, sc-7884, Santa Cruz Biotechnology); and rabbit polyclonal anti-IL-18 (H-173) antibody (1:1000, sc-7954, Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Cell Signaling Technology) was used as an internal reference. Blot bands were quantified via densitometry with ImageJ software (National Institutes of Health, Baltimore, MD, USA), and protein levels were expressed as the ratio of values of the detected protein bands to that of GAPDH bands.

The periodic acid-Schiff (PAS) staining was performed using the PAS staining kit (#395B-1KT; Sigma-Aldrich, St. Louis, MO, USA). For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA). For quantification of PAS and immunohistochemical staining, six areas from each section were analyzed using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, Version 6.3). Immunofluorescence staining of rat kidney sections with the β-catenin antibody (GTX101435; GeneTex, Irvine, CA, USA) was conducted according to the previously described procedure [49 (link)]. The mean fluorescence intensity of β-catenin in kidney sections was quantified using the CellSens software package Version 2.1 (Olympus Medical Systems, Tokyo, Japan).