Ab52954

Proteins from the liver homogenate and hepatic fractions were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, transferred to nitrocellulose membranes, and blocked for 1 h in Tris-Buffered Saline and Tween 20 (TBST) buffer (20 mmol/L Tris, pH 7.5, 500 mmol/L NaCl, 0.5% Tween 20) with 5% non-fat milk. Anti-TPH1 (ab52954, Abcam, Cambridge, U.K.) (1/3500 in TBST) or anti-MAOa (sc-20156, Santa Cruz Biotechnology, Inc, Santa Cruz, CA) (1/1000 in TBST) was added as primary antibody and incubated overnight at 4°C. As loading controls for liver homogenates, membranes were incubated with mouse anti-tubulin antibody (ab 56676 Abcam, UK) diluted 1/1000, for mitochondrial fractions was used as control protein of mitochondrial origin, rabbit anti-VDAC1/Porin antibody (ab15895; Abcam, UK) diluted 1/1000. After washing, membranes were incubated with the appropriate secondary antibodies conjugated to alkaline phosphatase (sc2315, Santa Cruz Biotechnology, Inc.)1/5000. Bands were revealed using the AP conjugate substrate kit (Bio-Rad, CA). Densitometric analysis was performed using the Image Lab Software (v 3.0, Bio-Rad, CA).

The entire small intestine samples (2 cm regions of the proximal, medial, and distal of the rat small intestine) were homogenized in lysis buffer with 1 mM phenylmethylsulfonyl fluoride and centrifuged at 15,000 × g for 15 min. The protein concentrations were measured by the BCA assay (BioTeke Corporation, Beijing, China). After denaturation, proteins were subjected to SDS-PAGE, blotted onto polyvinylidene difluoride membranes and blocked in Tris-buffered saline containing 0.1% Tween-20 with 5% nonfat milk for 2 h. Then, the membranes were incubated with antibody against TPH1 (1:500) (ab52954, Abcam), SERT (1:250) (ARG63804, Arigo) or glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (1:1000) (TA802519, Origen) overnight at 4°C. After overnight incubation, the membranes were washed in Tris-buffered saline containing 0.1% Tween-20 thrice and incubated with anti-rabbit IgG (1:10000) (ZB-5301, ZSGB-BIO, Beijing, China), anti-goat IgG (1:15000) (ZB-2306, ZSGB-BIO, Beijing, China), and anti-mouse IgG (1:10000) (ATA0011, ATgene Biotech, Chongqing, China) for 2 h each and then visualized by electrochemiluminescence detection (Fujifilm, Japan).

Beta nicotinamide mononucleotide (β-NMN) (BTO5) was purchased from BONTAC (Shenzhen, China). Nicotinamide phosphoribosyltransferase (Nampt) inhibitor (GMX1778) was from Selleck (Shanghai, China). Lomotil (Atropine-diphenoxylate) was purchased from Kangpu Pharmaceutical Ltd Co (Changzhou, China). Antibodies against serotonin transporter (ab181034), tryptophan hydroxylase (ab52954), Lgr5 (ab75850) and Nampt (ab45890) were purchased from Abcam (Cambridge, MA, USA). SR4 (sc-376158), p15/16 (sc-377412) and goat anti-mouse IgG-FITC (sc-2010) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). APC anti-mouse CD117 (c-kit) (135108) was purchased from BioLegend (San Diego, CA, USA). Proliferating cell nuclear antigen (2586), GAPDH (5174) and Senescence β-Galactosidase Staining Kit (9860) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse IgG (31430), goat anti-rabbit IgG (31460) and TRIzol Reagent (15596-018) were purchased from Invitrogen (Carlsbad, CA, USA). EvaGreen Supermix (172-5201AP) was purchased from Bio-Rad Laboratories Inc (Hercules, CA, USA). PrimeScript first strand cDNA Synthesis Kit (6110A) was purchased from TaKaRa Bio Inc (Dalian, China). Nuclear Fast Red solution (N3020) and all other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise stated.