Ab72608

Ice-cold radio-immuno-precipitation assay (RIPA) buffer was applied to extract the proteins from tissue homogenates, either from the urinary bladder or heart. Western blotting analysis was subsequently performed using our previous method [31 (link)]. The target antigens from the protein extracts were characterized using the primary antibodies specific for p-STAT3 (1:1000, phosphor-Y705, ab76315, Abcam), STAT3 (1:1000, ab68153, Abcam), TGR5 (1:1000, ab72608, Abcam), or β-actin (1:5000, A5316, Sigma-Aldrich, St. Louis, MO, USA). The bound primary antibodies were then hybridized to horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgGs (Calbiochem, San Diego, CA, USA), and the immunoreactive bands were developed with a chemiluminescence kit (Perkin Elmer, Waltham, MA, USA). Based on the optical densities of each band, p-STAT3 (88 kDa), STAT3 (88 kDa), TGR5 (35 kDa), or β-actin (43 kDa) was quantified using our previous method [31 (link)].

The liver and distal ileum was collected at 8 weeks post surgery, homogenized in lysis buffer and pelleted. Protein concentrations of the supernatant were analysed, and equivalent amounts of protein were loaded onto a polyacrylamide gel. Primary antibodies raised against TGR5 (ab72608; 1:1,000), CYP7A1 (ab65596, 1:1,000) were purchased from Abcam (Cambridge, MA). Goat anti-FXR (sc-1204; 1:1,000), rabbit anti-OATPβ (sc-135099; 1:500), was from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep anti-FGF15 (1:1,000) was purchased from R&D Systems (Minneapolis, MN). Rabbit anti-SHP2 was a gift from Dr Gen-Sheng Feng, the University of California San Diego, CA. The primary antibodies were diluted in Li-Cor blocking buffer and incubated at room temperature for 1 h. Goat anti-rabbit, donkey anti-goat, goat anti-mouse or donkey anti-sheep fluorescently labelled secondary antibodies (Li-Cor Inc., Lincoln, NE) were diluted 1:10,000 and incubated in blocking buffer at room temperature for 1 h with shaking. After three rinses with PBS solution, the membrane was scanned with the Odyssey Infrared Imaging System (Li-Cor Inc.).

Colonic tissues or RIN-14B cells were fabricated using RIPA buffer (Signaling Technology, Inc.). The protein concentration was examined by a BCA kit (Sigma-Aldrich; Merck KGaA). Total protein (30 μg/sample) was separated via 10% SDS-PAGE and to nitrocellulose membranes. 5% nonfat dried milk was used to block the membranes. The corresponding protein antibodies were as follows: TGR5 (Abcam, ab72608; 1/1000), transient receptor potential ankyrin 1 (TRPA1; Alomone Labs, ACC-037; 1/1000), phospholipase C (PLC)-γ1 (Abcam, ab76155; 1/5000), phosphatidylinositol 4,5-bisphosphate (PIP2; Santa Cruz, sc-53412; 1/200), Tph-1 (Abcam, ab228588; 1/1000), and β-actin (Boster, BM0627; 1/1000). Then, the membrane washing was performed with Tris-buffered saline/0.1% Tween (TBST) and incubated for 1.5 hours with an HRP goat antirabbit IgG (Abcam, ab6721). The band visualization was carried out using the ECL system (Affinity Biosciences, Cincinnati, Ohio, USA), and as an internal control, β-actin was used.

Brain samples were collected 24 h after MCAO. Western blotting was performed as described previously [35 (link)]. The proteins of right hemispheres were extracted by cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein (50 μg) were loaded and subjected to electrophoresis on an SDS-PAGE gel. After being transferred to a nitrocellulose membrane, the membranes were cut into different strips which covered an area with target protein according to molecular weight marker location and were blocked with 5% nonfat milk (Bio-Rad Laboratories, Irvine, CA, USA). The membrane was incubated with the primary antibody overnight at 4 °C. Primary antibodies included the following: anti-TGR5 (1:1000; ab72608; Abcam, Cambridge, MA,USA), anti-Pellino3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-caspase-8 (1:1000; ab25901; Abcam, Cambridge, MA,USA), anti-NLRP3 (1:500; NBP2-12446; NOVUS, CO, USA), anti-caspase-1 (1:1000; NBP1-45433; NOVUS, CO, USA), and IL-1β (1:1000; ab2105; Abcam, Cambridge, MA, USA). β-actin (1:1000, sc-58673, Santa Cruz, TX, USA) was used as an internal loading control. The secondary antibodies were all from Santa Cruz Biotechnology. Blot bands were quantified by densitometry using ImageJ software (ImageJ 1.4, NIH, USA).

The proteins were extracted for western blotting as we reported previously. Proteins (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes (Immobilon-P^SQ, Millipore, Billerica, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA), incubated with primary antibodies, rabbit anti-TGR5 (1:200; ab72608, Abcam, Cambridge, MA, USA) and mouse anti-tubulin (1:500; AT819, Beyotime, Shanghai, China), and incubated with secondary antibody conjugated to horseradish peroxidase (1:2000, Zhongshan Inc., Beijing, China). Immunolabeled proteins were detected by the ChemiDoc XRS + Image System (Bio-Rad Laboratories, Bay Street, CA).

Protein lysates were prepared by adding lysis buffer (50 mM TrisBase, 0.25 M NaCl, 5 mM EDTA, 1 % Triton X-100, and 4 mM NaF) containing protease inhibitor. Cell lysates were centrifuged at 15,000 g for 15 min at 4 °C. To obtain the membrane microdomain enriched samples the lysate was centrifuged at 200,000 g for 1 h (Beckman Ultracentrifuge Ti 70.1 Rotor) [61 (link)]. Western blot samples were denatured by heating to 37 °C in 50 mM dithiothreitol for 30 min and run on precast gels from Invitrogen. The membranes were blocked overnight at 4 °C in 0.5 % milk powder and 1 % BSA. Primary antibody for TGR5 (1:400 rabbit, Abcam ab72608) were added in blocking buffer for 1.5 h. The goat anti-rabbit secondary antibody conjugated to horse-radish peroxidase (1:2.500) was added in blocking buffer, for 1 h. EZ-ECL chemiluminescence detection kit for HRP (BI, Biological Industries) was added and blots were viewed on Fusion FX Vilber Lourmat.