Ecl plus chemiluminescence substrate

Cells were washed twice in cold phosphate buffered saline (PBS). Total protein was extracted using 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, Indianapolis, IN, USA). Protein concentration was determined using Bradford Reagent (Sigma-Aldrich, Milano, Italy). Total protein extracts (40 μg) were separated by 10 % SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated overnight with primary anti-Spred1 antibody diluted 1:1000 (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA), anti-Irak1 antibody diluted 1:250 (MBL International Corporation, Nagoya, Japan), and anti-IL-1β antibody diluted 1:1000 (Cell Signaling Technology, Beverly, MA, USA); subsequently they were incubated with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Immunoreactive proteins were visualized using ECL Plus chemiluminescence substrate (GE Healthcare, Pittsburgh, PA, USA). Membranes were incubated with anti β-actin diluted 1:10,000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as an endogenous control.

Cells were washed twice in cold PBS. Total protein was extracted using 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, Indianapolis, IN). Protein concentration was determined using Bradford Reagent (Sigma-Aldrich, Milano, Italy). Total protein extracts (40 μg) were separated by 10 % SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were incubated overnight with primary anti-SPRED-1 antibody (Thermo Scientific, Pierce Biotechnology, Rockford, IL) and subsequently incubated with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Immuno-reactive proteins were visualized using ECL Plus chemiluminescence substrate (GE Healthcare, Pittsburgh, PA). Membranes were incubated with anti β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) as an endogenous control.