Anti nuclear factor κb nf κb p65

Protein samples extracted from kidney tissues were loaded onto gradient polyacrylamide gels and then transferred onto nitrocellulose membranes. The membranes were probed with specific primary antibodies as follows: anti-NGAL (Santa Cruz Biotechnology), anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), anti-cleaved poly(ADP-ribose) polymerase-1 (PARP-1; Cell Signaling), anti-p53 (Cell Signaling), anti-Bax (Santa Cruz Biotechnology), anti-tumor necrosis factor-α (TNF-α; Abcam), anti-interleukin-6 (IL-6; Abcam), anti-nuclear factor-κB (NF-κB) p65 (Cell Signaling), anti-p-NF-κB p65 (Cell Signaling), anti-α-SMA (Sigma-Aldrich), anti-fibronectin (Abcam), anti-transforming growth factor-β (TGF-β; R&D Systems, Minneapolis, MN, USA), anti-p-Smad2/3 (Cell Signaling), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling) antibody. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Signals were detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using the ChemiDoc™ XRS+ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). The protein expression levels were normalized against GAPDH.

Liver tissues were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) with protease and phosphatase inhibitors (Roche, Indiana, United States). The prepared protein was separated by 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Temecula, CA, United States), and incubated with the primary antibody and secondary antibody, respectively. Anti-FXR, anti-FGF15, and anti-small heterodimer partner (SHP) were obtained from Abcam (Cambridge, United Kingdom), anti-cholesterol 7-alpha hydroxy-lase (CYP7A1) and anti-cytochrome P450, family 8, subfamily B, polypeptide 1 (CYP8B1) were purchased from Santa Cruz Biotechnology (Texas, CA, United States), anti-nuclear factor-κB (NF-κB) p65 and anti-NF-κB P-p65 were purchase from Cell Signaling Technology (Beverly, MA, United States). As an internal control, β-actin was purchased from HuaBio (Hangzhou, China). Anti-rabbit IgG and anti-mouse IgG was obtained from Cell Signaling Technology (Beverly, MA, United States). The bands were visualized by ECL chemiluminescence detection kit (Millipore, MA, United States), and quantified using the Tanon 5200 Chemiluminescent Imaging System (Tanon Science & Technology Inc., Shanghai, China).

Proteins were extracted from liver tissues with a lysis buffer (Sigma-Aldrich). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The membranes were incubated with one of the following primary antibodies: anti-cleaved caspase-3 (1:1000; Cell Signaling, Danvers, MA, USA), anti-poly(ADP-ribose) polymerase-1 (PARP-1; 1:1000; Cell Signaling) anti-tumor necrosis factor-α (TNF-α; 1:1000; Abcam), anti-IL-6 (1:1000; Abcam), anti-IκBα (1:1000; Cell Signaling), anti-p-IκBα (1:1000; Cell Signaling), anti-nuclear factor-κB (NF-κB) p65 (1:1000; Cell Signaling), anti-p-NF-κB p65 (1:1000; Cell Signaling), anti-transforming growth factor-β1 (TGF-β1; 1:1000; Abcam), anti-Smad2/3 (1:1000; Cell Signaling), anti-p-Smad2/3 (1:1000; Cell Signaling), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:3000; Cell Signaling). The membranes were washed and probed with a secondary antibody conjugated with horseradish peroxidase. The signal intensities were analyzed using the iBright™ CL1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).