Westernbreeze blocker diluent

NHEKs were cultured on slides (Lab-Tek, Rochester, NY, USA). These slides were then washed in phosphate-buffered saline (PBS), fixed with acetone for 10 min, and blocked using 10% bovine serum albumin (Roche Diagnostics, Basel, Switzerland) in PBS for 30 min for the staining of IL-37. Samples were incubated with primary anti-human IL-37 polyclonal rabbit antibody (1:100) (Abcam) or IgG rabbit polyclonal antibody (Abcam) in Western Breeze Blocker/Diluent (Thermo Fisher Scientific) overnight at 4°C. The slides were then washed with PBS before incubation with anti-rabbit secondary antibody (Alexa Fluor 546; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. After nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI), the slides were mounted with UltraCruz mounting medium (Santa Cruz Biotechnology, Dallas, TX, USA). All samples were analyzed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan).

NHEKs were incubated for 5 min in lysis buffer (Complete Lysis M; Roche Diagnostics, Rotkreuz, Switzerland). The lysate protein concentration was measured with a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein were dissolved in NuPage LDS sample buffer (Thermo Fisher Scientific) and 10% NuPage sample reducing agent (Invitrogen). Lysates were heated at 70 °C for 10 min and loaded and run on Bis-Tris Gel (Thermo Fisher Scientific) at 200 V for 20 min. The proteins were transferred to PVDF membrane (Merck Millipore, Burlington, MA, USA) and blocked in Western breeze blocker/diluent (Thermo Fisher Scientific). Membranes were probed with primary antibody or anti-β-actin antibody overnight at 4 °C. Anti-mouse and anti-rabbit horseradish peroxidase-conjugated IgG antibodies (Cell Signaling Technology) were used as secondary antibodies. Visualization of protein bands was accomplished with Super Signal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific) using the ChemiDoc Touch Imaging System (Bio-Rad).

NHEKs were incubated for 5 min in Complete Lysis-M (Roche Diagnostics, Rotkreuz, Switzerland) for western blotting analysis of IL-37, IL-33, IL-36γ, or AHR. The protein concentration of collected cell lysates was measured using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA), following the manufacturer’s protocol. Equal amounts of protein were mixed with 10% NuPage sample reducing agent 10× (Thermo Fisher Scientific) and 25% NuPage LDS sample buffer 4× (Thermo Fisher Scientific), and then heated at 70°C for 10 min. Next, the proteins were loaded and run on 4%–12% Bis-Tris Gel (Thermo Fisher Scientific) at 200 V for 20 min and transferred to a PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were blocked in WesternBreeze blocker/diluent (Thermo Fisher Scientific) for 30 min and then probed with primary antibody overnight at 4°C. They were subsequently incubated with anti-mouse horseradish peroxidase-conjugated IgG secondary antibody (Cell Signaling Technology) or anti-goat horseradish peroxidase-conjugated IgG secondary antibody (Promega Corporation, Madison, WI, USA). Protein bands were visualized with Super Signal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific) using the ChemiDoc Touch Imaging System (Bio-Rad).