Ab203043

Tissue lysates were prepared using Cell Lysis Buffer for Western and IP (Biotime Biotechnology, Xiamen, Fujian, PRC). Protein concentrations were detected using a Enhanced BCA Protein Assay Kit (Biotime Biotechnology). Equal amounts of proteins from each sample were subjected to SDS-PAGE followed by a transfer of proteins to nitrocellulose membranes. Membranes were blocked with a non-protein blocking solution (Sangon Biotech) and incubated with a primary antibody overnight at 4°C. After washing with TBST, membranes were incubated with a secondary antibody linked to Horseradish peroxidase (HRP). The blots were then developed with an ECL detection system as per the manufacturer's instructions. Rabbit anti-SGLT1 (ab14686), anti-PEPT1 (ab203043), anti-P47^phox (ab74095), anti-Nrf2 (ab62352), anti-Keap1 (ab139729), anti-iNOS (ab178945), anti-MR (ab229987), anti-p-IRF3 (Ser396)(ab138449), and anti-IRF3 (ab25950) monoclonal antibodies were obtained from Abcam. Rabbit anti-Arg (93668S), anti-pSTAT1 (Tyr701) (9167S), anti-STAT1 (9172T), anti-STAT6 (5397S), anti-STAT3 (30835S) monoclonal antibodies were obtained from CST (Shanghai, PRC). Mouse anti-β-actin monoclonal antibody was obtained from Biotime Biotechnology. The relative band density was determined using ImageJ software.

The total protein from hypothalamus and colon were separated by SDS-PAGE and then transferred to PVDF membrane. The membrane was soaked with skimmed milk powder to reduce the binding of non-specific antibodies. The membranes were then exposed to primary antibodies and secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson) or peroxidase-conjugated goat anti-mice IgG (115–035-003, Jackson) depending on the primary antibody). Protein was quantified with Lab image Software (BioRad, USA) and expressed as relative units to housekeeping proteins.
The primary antibodies are as follows: anti-free fatty acid receptor 2 (FFAR2; ABC299, Merck Millipore), anti-monocarboxylic acid transporter 1 (MCT1, ab93048, Abcam), anti-small peptide transporters (PEPT1, ab203043, Abcam), anti-GAPDH (A01020, Abbkine), and anti-β-tubulin (A01030HRP, Abbkine).