Anti actb antibody

Lysates from the cell culture were prepared using RIPA buffer (Wako Pure Chemical Industries). Cells were washed with cold PBS and incubated with RIPA buffer for 15 min on ice. Cell lysates were subsequently transferred into 1.5 ml tubes and centrifuged at 12,000 × g for 20 min at 4°C. Supernatants were transferred to a fresh tube and stored at –80°C before analysis. A total of 10 µg protein was loaded per lane and separated by 10% SDS-PAGE. The expression of pro-IL-1β and β-actin (ACTB) was analyzed by western blot. After transfer onto polyvinylidene fluoride membranes, nonspecific antibody binding was blocked for 1 h at room temperature using Immunoblock (DS Pharma Biomedical, Osaka, Japan). Then, membranes were incubated for 24 h at 4°C with anti-IL-1β antibody (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-ACTB antibody (1:10000, Sigma-Aldrich), followed by an incubation for 1 h with secondary antibody conjugated horseradish peroxidase (HRP; 1:1000, GE Healthcare, UK, Buckinghamshire, UK). Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (GE Healthcare) using ImageQuant LAS 4000 (GE Healthcare). The results represent at least 3 independent experiments. Quantitative analysis of bands was performed using Image J (National Institutes of Health, Bethesda, MD, USA).

Lysates from the cell culture were prepared using RIPA buffer (FUJIFILM Wako Pure Chemical). The expression of NLRP3, and ACTB were analyzed using SDS-PAGE. After transfer onto
polyvinylidene fluoride membranes, nonspecific antibody binding was blocked for 1 h at room temperature using Immunoblock (DS Pharma Biomedical, Osaka, Japan). Then, membranes were
incubated for 24 h at 4°C with anti-NLRP3 antibody (1:1000, R&D) and anti-ACTB antibody (1:10000, Sigma-Aldrich), followed by an incubation for 1 h with secondary antibody
conjugated horseradish peroxidase (HRP; 1:1000, GE Healthcare, UK, Buckinghamshire, UK). Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (GE Healthcare)
using ImageQuant LAS 4000 (GE Healthcare). The results represent at least 3 independent experiments. Quantitative analysis of bands was performed using Image J (National Institutes
of Health, Bethesda, MD, USA).

To evaluate the expression of MMPs, western blot analysis was performed. The cells were lysed with PRO-PREP™ solution (Intron Biotechnology, Sungnam, Korea), and equal amounts of cell extracts were analyzed using 4% to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and blocked with 4% nonfat milk in Tris-buffered saline and 0.1% Tween 20 at room temperature. Primary antibodies (MMP 1–2: Cell Signaling, Beverly, MA, USA; MMP9: Santa Cruz Biotechnology), secondary antibodies (Enzo Life Sciences, Minneapolis, MN, USA), and Amersham ECL Select Western blot detection reagent (GE Healthcare Life Sciences, Buckinghamshire, UK) were used. Anti-ACTB antibody (Sigma-Aldrich) was used for each probing. ImageJ software (version 1.52a; National Institutes of Health, Bethesda, MD, USA) was used for band intensity quantification. In order to calculate relative protein expression ratios, the protein expressions in the treated cells were divided by those of the control cells.