Nonfat milk

After 24 h, cells were harvested and dissolved in lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerin, and 200 mM dithiothreitol). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Samples were mixed with Laemmle’s loading buffer and boiled for 7 min. Equal amounts (20–40 µg) of protein extracts were loaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel) at 100 mV, followed by blotting onto nitrocellulose membranes for 1 h 50 min at 110 V on ice. After transfer, nitrocellulose membranes stained with Ponceau S. Membranes were blocked for 1 h with 5% nonfat milk (AppliChemGmbH, Darmstadt, Germany, A0830) in tris-buffered saline (TBS) at room temperature, probed overnight with the appropriate primary antibodies (1:3000–1:5000) and followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, anti-mouse 7076S or anti-rabbit 7074S, 1:4000) for 1 h with 2.5% nonfat milk in Tris-buffered saline (TBS) at room temperature. Detection was visualized with ECL (Amersham Biosciences, Piscataway, NJ, USA) and ChemiDoc MP Imaging System (BioRad). The quantitative analysis of Western blot data was accomplished using ImageJ Software (NIH, Bethesda, Rockville, MD, USA).

Protein lysates from cells or liver tissues were separated on a 4%–12% NuPAGE Bis‐Tris protein gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Chicago, IL, USA). Blots were blocked with 5% nonfat milk (Nestle, Jacksonville, IL, USA), then incubated with primary antibodies in Tris‐buffered saline containing 0.1% Tween 20 (Sigma) and 5% nonfat milk, followed by HRP‐conjugated anti‐rabbit antibody (Cell Signaling, Danvers, MA, USA, 7074S, at 1:3000 dilution) or anti‐mouse antibodies (Cell Signaling, 7076S, at 1:2500 dilution). Primary antibodies used in this study were anti‐NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000); anti‐ASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000); anti‐Casp1 (p10) mouse antibody (Adipogen, AG20B0044C100, 1:1000); anti‐tubulin rabbit polyantibody (Santa Cruz, Dallas, TX, USA, SC‐5286, 1:200); anti‐Nek7 rabbit antibody (Abcam, Cambridge, MA, USA, ab133514, 1:10000); and anti‐IL‐1β goat antibody (R&D systems, Minneapolis, MN, USA, AF401NA, 1:2000).

One million BM-DCs were stimulated with 1 or 4 μg/ml Man- and Gal-LPS, respectively, for the indicated time period. Cells were then processed as described previously with adaptation of lysis buffer volume to 75 μl (69 (link)). Cell lysate equivalent to two hundred thousand cells (15 μl) was subjected to SDS-PAGE using a 4–15% gradient TGX minigel (Bio-Rad) for 90 min at 100 V. Proteins were transferred onto nitrocellulose membrane (GE Healthcare) at 100 V for 30 min. The membranes were blocked with 5% nonfat milk (Lonza) in PBS containing 0.05% Tween 20 (PBS-T) for 1 h at 25 °C. The blocked membranes were washed four times by incubating in PBS-T for 5 min each. Membranes were then incubated with the primary antibodies in PBS containing 1% bovine serum albumin for 1 h at 25 °C at a dilution of 1:1000 for Syk, phospho-Syk, IκB, and β-actin and 1:5000 for p38 and phospho-p38. Membranes were washed as above and probed with anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling) in 5% nonfat milk in PBS-T at a dilution of 1:3000 for 1 h at 25 °C. Membranes were washed as above and incubated with the ECL detection reagent (GE Healthcare) and imaged using Fluorochem E (ProteinSimple).