Sc 13063

Ten-day and 8-week-old WT or FXR Ko mice were sacrificed and intracardially perfused. The spleens, spinal cords and brains were removed and fixed in 4% PFA overnight. Paraffin sections (4 μm) were pretreated with citrate buffer (pH 6) and stained using an automated immunostainer (AutostainerLink 48, Dako). Primary antibodies were specific to FXR (NR1H4, rabbit, Abcam ab28676 1:200; or Santa Cruz sc-13063, 1:50), NogoA (mouse, 11c7, a generous gift from M.E. Schwab, Brain Research Institute, University of Zürich and Department of Biology, Swiss Federal Institute of Technology Zürich, Switzerland, 1:15,000) and Olig2 (rabbit, 18953, IBL, 1:150 and mouse, 387M-16, Medac, 1:200). Numbers of oligodendroglial cells were quantified in a blinded fashion in the corpus callosum, cerebellum and spinal cord in standardized microscopic fields of 10,000 μm² each defined by an ocular morphometric grid.

Tissue sections were stained with hematoxylin and eosin (HE), Periodic acid-Schiff’s (PAS) and Masson’s trichrome stains using standard protocols. The mesangial area was determined from the PAS stained sections and the fibrotic area from the Masson’s trichrome stained sections as previously described^{28 (link)}. The antibodies used in this study were those against FXR (sc-13063, Santa Cruz), Grp78 (ab21685, Abcam), Chop (ab59396, Abcam), 4-hydroxynonenal (4-HNE, ab46545, Abcam), Kim-1 (ab78494, Abcam), 8-oxo-2′-deoxyguanosine (8-oxo-dG, ab64548, Abcam), SDHA (#11998, CellSignaling, Danvers, MA, USA), tumor necrosis factor (TNF, ab6671, Abcam), CD4 (sc-7219, Santa Cruz, Dallas, TX, USA), aSMA (NBP1-30894, Novus Biologicals, Littleton, CO, USA) and Collagen I (ColI, NB600-408, Novus Biologicals). TUNEL staining on kidney paraffin sections was performed with an ApopTag kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions.

RAW 264.7 cells with different treatments were homogenized in RIPA buffer with protease and phosphatase inhibitors. The nuclear and cytoplasmic extraction was conducted under the protocol of the Nuclear and Cytoplasmic Extraction Kit (CW0199, CWBIO, China). The protein extracts were then separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was incubated with antibodies against Nr1h4 (sc-13063, Santa-Cruz, 1: 1000), GAPDH (Cat#2118, Cell Signaling, 1:1000) and LaminB (GTX103292, GeneTex, 1:1000) overnight at 4°C.

Tissue protein extracts underwent gel electrophoresis and proteins were then transferred to membranes (Arsenijevic et al., 2015 (link)). Membranes were pre-incubated with 1% casein (Vectorlab) for 2 h and then rinsed. Membranes were then incubated 2 h with one of the following primary antibodies—uncoupling protein-1 (UCP1) dilution 1/5000 (cat. no. UCP11, Alpha Diagnostics), SIRT1 dilution 1/200 (sc-19857, Santa Cruz), farnesoid x receptor (FXR) dilution 1/200 (sc-13063, Santa Cruz), and beta-actin dilution 1/1000 (Cat No. 4970–Cell Signaling). Secondary antibody LI-COR anti-rabbit (dilution 1/15000) or anti-goat (dilution 1/15000) were used to detect bands (de Bilbao et al., 2009 (link)). The signals were visualized with the use of Odyssey Infrared Imaging System (Li-Cor Biosciences, Bad Homburg, Germany).

The HASMCs and rat tissues were lysed with RIPA buffer containing complete protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were measured using a BCA protein assay kit (Interchim, Montluçon, France). The samples were separated by 10% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the resulting protein bands were transferred onto PVDF membranes. After being blocked, the membranes were incubated with the following primary antibodies overnight at 4 °C: NF-κB antibodies (1:1000, ab32536, Abcam), TNF-α antibodies (1:1000, ab6671, Abcam), FXR antibodies (1:500, sc-13063, Santa Cruz Biotechnology), TAK1 antibodies (1:1000, ab109526, Abcam), p-TAK1 antibodies (1:1000, ab109404, Abcam), TAB1 antibodies (1:1000, ab227210, Abcam), and Phospho-IκBα antibodies (Ser32/36) (1:1000, #9246, Cell Signaling Technology). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours. The protein bands were detected using an ECL Substrate (Thermo Fisher Scientific), and the quantification of the average densities of the protein bands was performed using the Image-Pro Plus 4.5 software.