Fusion fx7 device

The CYP51A, CYP51B, and CYP51C genes were amplified using previously described primers [27 (link)]. The reaction mixtures contained 0.2 µM of each primer, 25 µL of Taq’Ozyme Purple Mix 2 (Ozyme, Saint-Cyr-l’École, France), and 5 µL of gDNA in a final volume of 50 µL. Samples were amplified using a CFX96 Real-Time PCR System (BioRad, Marnes-la-Coquette, France) and the following cycling protocol: one initial cycle of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C, and 2 min at 72 °C, with one final cycle of 5 min at 72 °C. PCR quality was assessed by 1.5% agarose gel electrophoresis (120 V, 20 min) and visualization using the Fusion Fx7 device (Vilber Lourmat, Eberhardzell, Germany). PCR product sequencing was performed using the Sanger method by Eurofins Genomics (Ebersberg, Germany) with the same primers.

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.