Blotting buffer

The protein concentrations of the cell lysate were determined using a DC Protein Assay Kit (Bio-Rad). To detect the expression of IL-37 in IL-37sgRNA and TFneg cells, 50 μg total protein from cell lysates was resolved in 12% Bis/Tris gels (Novex, Life Technologies) in NuPage running buffer (Novex, Life Technologies) and transferred to nitrocellulose membranes in blotting buffer (Bio-Rad). After blocking in 5% bovine serum albumin (BSA, Carl Roth GmbH, Karlsruhe, Germany), nitrocellulose membranes were probed overnight at 4 °C using 3 µg/ml rabbit polyclonal anti-IL-37b (Novus Biologicals, Cambridge, UK). Rabbit polyclonal anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas) at a 1:15,000 dilution was used as a loading control. Blots were then incubated with a horseradish peroxidase-conjugated secondary anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) and visualized using a Luminata Forte Western HRP substrate (Merck Millipore, Billerica, MA, USA). The membranes were exposed to chemiluminescence using an Odyssey Fc (LI-COR Biosciences, Lincoln, NE, USA). Densitometric analysis was performed using version 3.1 of the Image Studio software package (LI-COR). The calculations were performed based on density of the IL-37 and GAPDH bands, respectively, as detected by the software, and the results were normalized based on IL-37/GAPDH.