Ab129089

Mouse liver tissues or macrophages were lysed using RIPA lysis buffer (beyotime) to obtain the protein sample. After the protein concentration was measured using a BCA kit (beyotime), certain volume of protein was mixed with loading buffer (beyotime) for boiling water bath for 3 minutes for degeneration. The proteins were subjected to electrophoresis at 80 V for 30 minutes and then at 120 V for 1–2 hours. Membrane was transferred at a current of 300 mA for 60 minutes and then washed in washing buffer for 1–2 minutes before blocking at room temperature for 1 hou or at 4℃ for overnight. The membranes were incubated with one of the primary antibodies against GAPDH (ab181602, 1:10 000), ApoM (ab85695, 1:1000), FXR1 (ab129089, 1:1000), ABCA1 (ab18180, 1:200), ABCG1 (ab52617, 1:1000) and SR‐BI (ab217318, 1:2000) (Abcam) at shaking bed at room temperature for 1 hour before washing for 3 × 10 minutes. After that, the membranes were incubated with horseradish peroxidase–labelled goat anti‐rabbit IgG (1:5000; Beijing ComWin Biotech Co., Ltd) for 1 hour and washed for 3 × 10 minutes. The membranes were observed under a chemiluminescence imaging system (Bio‐rad) after development solution was added.

Conformation-dependent amyloid-specific OC antibodies (AB2286, Merck, Burlington, MA, USA) were used at a dilution of 1:400, and the primary antibodies anti-FXR1 (ab129089, Abcam, Cambridge, UK) were used at a dilution of 1:600. Cryosections were pretreated as described earlier [5 (link)] and incubated with primary antibodies at +4 °C, overnight. After washing to remove unbound antibodies, the sections were incubated with anti-rabbit secondary antibodies conjugated with Alexa Fluor 647 (ab150075, Abcam, Cambridge, UK) at a dilution of 1:1000 for an hour at +37 °C. To visualize nuclei, the slides were counterstained with DAPI. Histological staining of amyloids was performed using 0.1 mg/mL CR solution in 50% ethanol or using 1% Thioflavin S solution in 70% ethanol. Visualization of the results of immunohistochemical analysis and staining with amyloid-specific dyes was performed with a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) and “Leica Application Suite X 3.3.0.16799” software. To quantify the colocalization of FXR1 with CR red or Thioflavin S, Pearson’s correlation coefficient was calculated using the coloc2 plugin of FIJI software (http://fiji.sc/Fiji, accessed on 23 February 2020). At least 100 zones on preparations of each animal studied were analyzed to assess the colocalization of CR or Thioflavin S with the FXR1 protein.

HB (collection in Sichuan, 20042901), ZZ (collection in Fujian, 210303), DH (collection in Gansu, 20201211), and MX (collection in Jiangsu, 21032501) were obtained from Simcare Ltd. Primary antibodies against ZO-1 (21773-1-AP, 1:1000), occludin (13409-1-AP, 1:1000), claudin-1 (13050-1-AP, 1:1000), and the second antibody of beta-actin (20536-1-AP, 1:10000) were purchased from Proteintech Group Inc. Primary antibodies against NTCP (ab131084, 1:1000), BSEP (ab155421, 1:1000), CYP7A1 (ab234982, 1:1000), MRP2 (ab172630, 1:1000), and FXR1 (ab129089, 1:1000) were obtained from Abcam Inc. ANIT (N106389), UDCA(U110695), and olive oil (O108685) were purchased from the Aladdin Chemical Reagent Co. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (reference D4540) (St-Louis, MO, USA). The TRIzol total RNA extraction kit was obtained from Life Technologies.