Nf κb p65 sc 8008

RIPA buffer (150 mMNaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA, pH 8.0) containing a protease inhibitor cocktail (Roche Applied Science, USA) and a phosphatase (Sigma-Aldrich) inhibitor cocktail was used to obtain the cell lysates. Protein concentration was determined using Bradford Reagent (Sigma-Aldrich, Milano, Italy). Total protein extracts (25 μg) were separated by SDS-PAGE and then transferred to a nitrocellulose membrane using the Trans-Blot Turbo™ Transfer system (Bio-Rad). Membranes were then blocked for 1 h at room temperature (RT) in TBS with 0.1% of Tween-20 containing 5% non-fat dried milk, and subsequently incubated overnight at 4 °C with the primary antibodies of interest. All primary antibodies were probed with a secondary horseradish peroxidase (HRP)-conjugated antibody (Vector, USA). Proteins were visualized by ECL and the chemiluminescent signaling acquired using ChemiDoc XRS + System (Bio-RadLaboratories, Hercules, CA, USA) and analyzed using Image J software (Version 1.50i, National Institute of Health, Bethesda, MD, USA).
The primary antibodies used were Caspase-3 (CAS3) (# 9664) (Cell Signaling Tecnologies), Phospho-NF-κB p65 (# 5970), NF-κB p65 (Sc-8008) (Santa Cruz Biotechnologies) Paraoxonase 2 (PON2) (# SAB2700275). Actin, vinculin, and glyceraldehyde-3-phosphate dehydrogenase (GADPH) were used as normalizers.

Western blot analysis was performed as previously described [16 (link)]. Briefly, whole-cell lysates were prepared in lysis buffer (Cell Signaling Technology, MA, USA). Samples containing 30 μg of protein were resolved by electrophoresis gels and transferred to a nitrocellulose membrane. After blocking for 1h with 5% skim milk in PBST, membranes were incubated overnight at 4 °C with anti-MMP-2 (MAB902, R&D Systems; Minneapolis, MN, USA), NF-κB p65 (sc-8008, Santa Cruz Biotechnology, Dallas, TX, USA), p38 (D13E1-Cell Signaling Technology, MA, USA), phospho–p38 (D3F9-Cell Signaling Technology, MA, USA), and anti-β-actin (Millipore Corporation, Billerica, MA, USA) primary antibodies. Membranes were then probed with goat anti-mouse IgG-HRP (31430, Thermo Scientific) secondary antibody at room temperature (RT) for 1 h. Detection of the immunoreactive bands was performed using the Clarity Western ECL Substrate (BioRad). β-actin served as a loading control. Densitometric analysis was performed using Image J.

Monosodium urate (MSU) crystals were obtained from InvivoGen (Carlsbad, CA, USA). Phorbol 12-myristate 13-acetate (PMA), colchicine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO; ≥99.5%) were bought from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for IL-1β (12242) and NLRP3 (15101) were purchased from Cell Signaling Technology (USA). Antibodies against caspase-1 (sc-56036), phospho-IκB-α (sc-52943) and NF-κB p65 (sc-8008) were from Santa Cruz Biotechnology (Dallas, TX, USA) and β-actin antibody was from Abcam (Cambridge, UK). Peroxidase-conjugated AffiniPure goat anti-mouse IgG (115-035-003), and anti-rabbit IgG HRP-linked antibody (7074) as secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA) and Cell Signaling Technology, respectively.