Sodium dodecyl sulphate polyacrylamide gel electrophoresis

Radioimmunoprecipitation buffer (Thermo Fisher Scientific, USA) was used to lyse the cells. Cell lysate containing 50 μg of total protein was transferred onto a polyvinylidene fluoride membrane (Millipore, USA) after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific, USA). The membrane and primary antibody were incubated at 4°C overnight. The protein bands were then rinsed with Tris buffer saline plus Tween (TBST) buffer 3 times, 10 min each. Next, the membrane and secondary antibody were incubated at room temperature for 2 h. Chemiluminescence substrate (Thermo Fisher Scientific, USA) was added to observe the protein bands.
The primary antibodies including Anti-FXR (ab129089, diluted at 1 : 1000), anti-DHRS9 (ab126074, diluted at 1 : 1000), anti-ATP5D (ab97491, diluted at 1 : 1000), anti-ATP5E (Cat #PA5-104424, diluted at 1 : 1000), anti-NDUFA3 (H00004696-K, diluted at 1 : 1000), and anti-GAPDH (ab9485, diluted at 1 : 2500) were all rabbit-derived antibodies. Anti-FXR, anti-DHRS9, anti-ATP5D, and anti-GAPDH were from Abcam (UK). Anti-ATP5E antibody was from Thermo Scientific (USA). The anti-NDUFA3 antibody was bought from Abnova (China). Goat anti-rabbit IgG H&L (HRP) antibody (Abcam, ab6721, diluted at 1 : 2000, UK) served as the secondary antibody.

Cells were lysed in a lysis buffer (Cell Signaling Technology). The protein concentration in the lysates was measured by BCA protein assay (TaKaRa, Kusatsu, Shiga, Japan). Equal amounts of samples (15 μg) were fractionated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Thermo Fisher) and the proteins were transferred onto PVDF membranes. After blocking, the membranes were incubated overnight with a primary antibody, followed by a horseradish peroxidase-conjugated secondary antibody. Target proteins were visualized by ImmunoStar LD (Wako, Osaka, Japan) and the membranes were scanned using a C-DiGit Blot scanner (LI-COR Biotechnology, Lincoln, NE). The blot images were semi-quantitated with Image Studio software. The antibodies used for Western blotting are listed in Table 3.

Livers were extracted in a lysis buffer (Cell Signaling Technology), and cleared supernatants were stored at −80 °C until use. Protein concentration in the lysates was measured by protein-dye binding assay (Bio-Rad Laboratories, Inc). Equal amounts (15 μg) of samples were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane. After overnight incubation with the appropriate primary antibody, the membrane was incubated with horseradish peroxidase-conjugated rabbit IgG antibody (Santa Cruz Biotechnology) and the presence of each protein was visualized with enhanced chemiluminescence detection reagents (ImmunoStar LD; Wako, Osaka, Japan). Blots were photographed and analysed with Image Studio software.