Imagequant las 4000 mini imager analysis

Mouse liver specimens were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4; 1% Triton X-100; 25 mM HEPES; 150 mM NaCl; 0,2% SDS; 5 mM MgCl₂; 1 mM Na₃VO₄; 1 mM NaF) containing protease inhibitors (Roche, #04 693 132 001) using an Ultra-Turrax® homogenizer. After 40 min in ice, samples were centrifuged at 15,000 g. Proteins from diluted supernatant were assayed with the Bradford method (BioRad). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blocked with non-fat milk in TBS (20 mM Tris, 137 mM NaCl) during 1–2 h and incubated overnight at 4 °C with anti-cleaved caspase-3, anti-RIPK1 (Cell Signaling, 3493) anti-actin (Sigma A3854), anti-phospho-Thr183/Tyr185-JNK (Cell Signaling, 9251) or anti-JNK (Calbiochem, 559304) primary antibodies, and then with secondary goat anti-rabbit immunoglobulins/HRP (Dako, P0448). Protein-antibody complexes were revealed by enhanced chemiluminescence (Millipore) and ImageQuant LAS-4000 mini imager analysis (GE-Healthcare). The Multi Gauge software was used for signal quantifications and data was expressed as levels relative to signals detected in PBS controls. Cleaved caspase-3 expression levels or JNK phosphorylation status were respectively normalized on β-actin expression levels or on total JNK expression levels.

Primary hepatocytes and mouse liver specimens were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4; 1% Triton X-100; 25 mM HEPES; 150 mM NaCl; 0,2% SDS; 5 mM MgCl₂; 1 mM Na₃VO₄; 1 mM NaF and proteases inhibitors (Roche, #04 693 132 001)), and liver were crushed with UltraTurax. After 40 min in ice, samples were centrifuged at 13 000 g. Proteins from supernatant were assayed with the Bradford method (Bio-Rad). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blocked with non-fat milk or BSA 3–5% in TBS (20 mM Tris, 137 mM NaCl) during 1–2 h and incubated overnight with primary antibody anti-cleaved-caspase-3, anti-RIPK1 (Cell Signaling) anti-actin (Sigma A3854), anti-TRAF2 (Santa Cruz Biotechnology, sc-876), anti-phospho-IκBα (Cell Signaling, #2859) or anti-IκBα (Santa Cruz Biotechnology, sc-371) at 4 °C, and then with secondary goat anti-rabbit immunoglobulins/HRP (Dako, P0448). Protein–antibody complexes were revealed by enhanced chemiluminescence (Millipore, Billerica, MA, USA) and ImageQuant LAS-4000 mini imager analysis (GE Healthcare, Chicago, IL, USA).