Ab75610

Crypts were homogenized in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) in the presence of a protease inhibitor cocktail (Nacalai Tesque) using a Nippi Biomasher (Nippi, Tokyo, Japan) for 1 h at 4 °C. Homogenized crypts were centrifuged at 20,000× g for 30 min to obtain supernatants. Protein concentrations in the supernatants were measured using a BCA protein assay kit (Thermo Fisher Scientific). Samples, including 10 mg of protein and 25 or 50 ng of mouse kidney lysate (positive control), were separated on an SDS-PAGE, following which proteins were transferred to nitrocellulose membranes. The membrane was blocked with StabilGuard (SurModics, Eden Prairie, MN, USA) for 1 h at 25 °C and then incubated at 4 °C overnight with 1 μg/mL anti-FFAR3/GPR41 (ab236654; Abcam, Cambridge, UK), anti-FFAR2/GPR43 (ABC299; Merck Millipore, Darmstadt, Germany), and anti-LAT2/Slc7a8 antibody (ab75610; Abcam) antibodies. After the membranes were washed, they were incubated for 1 h at 25 °C with goat anti-rat IgG-HRP (Imgenex, San Diego, CA, USA). After another wash, the proteins were detected using a chemiluminescent substrate (Chemi-Lumi One, Nacalai Tesque).

Tissues were homogenized in lysis buffer (P0013B, Beyotime, Haimen, China) containing protease inhibitor cocktail (P0013B, Beyotime, Haimen, China). The homogenates were centrifuged (4 °C, 15871 rcf, 20 min), and the supernatant was collected. Approximately 20 μg of proteins was loaded on a Tris-glycine gel. Proteins were transferred onto PVDF membranes, blocked, and stained overnight using the following primary antibodies: anti-CHD8 (1:1000, ab114126, Abcam), HRP-conjugated anti-GAPDH (1:5000, BE0034, Easybio, Beijing, China), anti-SLC6A19 (1:5000, ab180516, Abcam), and anti-SLC7A8 (1:5000, ab75610, Abcam). Membranes were washed and stained with HRP-conjugated secondary antibodies (1:5000, BE0101 and BE0102, Easybio, Beijing, China) before washing and development with enhanced chemiluminescence (ECL) substrate for Western blotting (BE6706, Easybio, Beijing, China).

Approximately 40 μg protein per sample was separated on 12% SDS (sodium dodecyl sulfate) polyacrylamide gels. Proteins were transferred onto 0.45 μm PVDF membranes (IPVH00010; Millipore, Boston, Massachusetts, USA) and blocked with blocking buffer (Beyotime, Jiangsu, China). The membranes were incubated with primary antibodies to SCP2 (non-specific lipid-transfer protein 2, ab140126; Abcam, Cambridge, MA, USA), IDH2 (isocitrate dehydrogenase 2; ab131263, Abcam), SLC7A8 [(solute carrier family 7 (amino acid transporter, L system), member 8, ab75610, Abcam)], COL4A2 (collagen, type IV, alpha 2, sc-70,243, Santa Cruz biotechnology; Cambridge, MA, USA), and β-actin (Beyotime). After washing with TBST [tris-buffered saline containing 0.02% (v/v) Tween-20] three times, the membranes were incubated with goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies conjugated with horseradish peroxidase (Beyotime), incubated with ECL (electrochemiluminescence) Western Blotting Substrate Kits (Beyotime), and finally visualized with a Kodak Image Station 2000MM (Kodak Molecular Imaging Systems, New Haven, USA). The relative intensities of bands were calculated with ImagePro Plus 6.0 software (Media Cybernetics, Washington, MD, USA) using β-actin as the reference protein.