Bio 2d software

Human primary fibroblasts were lysed in cell lysis buffer (100 mM Pipes, 1 mM MgCl₂, 2 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, protease inhibitor). The protein concentrations of cell lysates were determined using colorimetric assay. For the first dimension step, 50 μg of total protein was loaded onto immobilized pH gradient strips (11 cm, pH 4-7) (BioRad, USA) via passive rehydration. IPG strips were then subjected to isoelectric focusing on a Protean isoelectric focusing cell (BioRad, USA). For the second dimension, IPG strips were applied onto SDS-polyacrylamide gels. Gel imaging and spot analysis were performed using Bio 2D-Software (Vilber Lourmat).

Primary hepatocytes were lysed using a lysis buffer (5 M NaCl, 1 M Tris, pH = 8, 10% Triton-X 100), sonicated for 5 s and centrifuged at a speed of 14,000× g for 10 min (temperature: 4 °C). Supernatants (35–40 µg of protein) were diluted with a loading buffer (4× Laemmli Sample buffer, Bio-Rad, USA), denatured at 95 °C for 10 min, and separated by SDS-PAGE electrophoresis (10%). Proteins were transferred to a nitrocellulose membrane, blocked in 5% BSA in TTBS for 1.5 h and then incubated overnight at 4 °C with primary antibodies anti phospho-NF-κB p65 (Ser536) (dilution, 1:2000 v/v), anti NF-κB p65 (dilution, 1:3500 v/v), anti IκB-α (dilution, 1:3500 v/v), anti phospo-IκB-α (Ser132) (dilution, 1:1500 v/v), anti IKKβ (dilution, 1:3500 v/v), anti phospho-IKKα/β (Ser176/180) (dilution, 1:1500 v/v), as well as anti β-actin (dilution, 1:5000 v/v) as a loading control (all antibodies were from Cell Signaling Technology, Danvers, MA, USA). After being washed in TTBS buffer, membranes were incubated with swine anti-rabbit IgG-HRP secondary antibody (Dako, Glostrup, Denmark) and visualized using an ECL kit (LumiGLO^®, Cell Signaling Technology). A Fusion Fx7 device and Bio-2D software (Vilber Lourmat, Collegien, France) were used to quantify the signals. Results were normalized to β-actin.