Anti il 18

The lung tissues were homogenized using a homogenizer with RIPA buffer containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor PhosSTOP (Roche). Total protein was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% nonfat milk and then processed with primary antibodies: anti-p-Akt/Akt (1:1000), anti-NLRP3 (1:1000) and anti-ASC (1:1000) (cell signaling technology Inc., Beverly, MA, USA), anti-caspase-1 (1:500), anti-IL-18 (1:500) and anti-IL-1β (1:500) (Santa Cruz Biotechnology, CA, USA), and anti-GAPDH (1:2000) (KANGCHEN Biotech, Shanghai, China). All of the membranes were subsequently incubated with HRP-conjugated anti-rabbit IgG (Promega, Madison, WI, USA), polyclonal rabbit anti-mouse IgG (Dako, Copenhagen, Denmark), and all of the blots were detected via enhanced chemiluminescence (ECL; Thermo Scientific). For quantitative analysis, the intensity of protein bands was determined using Image J 1.38 × software (NIH, Bethesda, MD, 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.

Western blot analyses on samples from tongue, lung, and spleen metastases was performed as described previously [22 (link)]. The membranes were incubated with primary antibodies: anti-NLRP3 (sc-34411, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-ASC (sc-22514, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-IL-1β (sc-32294, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-IL-18 (sc-80051, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-N-cadherin (sc-393933, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), anti-E-cadherin (sc-8426, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), anti-MMP2 (sc-13595, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), anti-MMP9 (sc-393859, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-βactin for cytosolic fraction (1:500; Santa Cruz Biotechnology; Dallas, TX, USA. sc-8432). Signals were perceived with an enhanced chemiluminescence (ECL) detection system mixture according to the manufacturer’s instructions (Thermo Fisher, Waltham, MA, USA).