Sc 13083

Following treatment with ellagic acid, liver tissue samples were acquired and homogenized for 10 sec at 12,000 × g for 10 min at 4°C. Total protein was measured using a BCA assay reagent (Beyotime Institute of Biotechnology), and then 50 µg protein was subjected to 10–12% SDS-PAGE and electrotransferred onto a polyvinylidene difluoride membrane (BD Biosciences, San Jose, CA, USA). The membrane was blocked in 5% non-fat milk in phosphate-buffered saline (PBS; pH 7.4) for 2 h, and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-inducible nitric oxide synthase (iNOS; 1:400; sc-649; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-vascular endothelial growth factor (VEGF; 1:500; sc-13083; Santa Cruz Biotechnology, Inc.), anti-VEGF receptor 2 (VEGFR2; 1:3,000; ab11939; Abcam, Cambridge, UK) and β-actin (1:500; sc-7210; Santa Cruz Biotechnology, Inc.). Next, the membranes were washed with PBS and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:5,000; 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 2 h. Protein expression in the samples was detected by Amersham ECL Prime western blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA) and analyzed using AlphaEase FC (FluorChem FC2) software (Cell Biosciences Inc., Santa Clara, CA, USA).

Stomachs were prepared, stained, and imaged using methods modified from Ramsey et al.⁵⁷ (link) The primary antibodies used for immunostaining were goat anti–vascular endothelial growth factor B (1:100, sc-13083; Santa Cruz Biotechnology, Dallas, TX), anti-CD44v9 (1:10,000, LKGM002; Cosmo Bio, Carlsbad, CA), and goat anti-GIF (1:10,000; a gift from David Alpers, Washington University in St. Louis). After washing, sections were incubated with secondary antibodies and GS-II lectin (1:500, L21415; ThermoFisher, Waltham, MA). Sections were washed, stained with Hoechst (62249; ThermoFisher) 1:20,000 in PBS, and mounted in ProLong Gold Antifade mountant (P36934; ThermoFisher).

Stomachs were prepared, stained, and imaged using methods modified from Ramsey et al.³⁵ (link) The primary antibodies used for immunostaining were as follows: rabbit anti-IL-17RA (1:100, bs-2606R; BIOSS, Woburn, MA), goat anti–vascular endothelial growth factor B (VEGF-B) (1:100, sc-13083; Santa Cruz Biotechnology, Dallas, TX), and anti-CD44v9 (1:10,000, LKGM002; Cosmo Bio, Carlsbad, CA). Secondary antibody labeling was as described. For activated caspase-3 immunohistochemistry, a Cell Signaling Technologies (Danvers, MA) SignalStain Apoptosis Kit (12692S) was used according to the manufacturer’s protocol. For terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, an In Situ Death Detection Kit (11684795910; Millipore-Sigma, St. Louis, MO) was used according to the manufacturer’s specifications. Slides of glands for immunofluorescence were generated using a Cytospin 4 centrifuge (A78300003; ThermoFisher, Waltham, MA). Cells were fixed on the slide in 4% paraformaldehyde for 20 minutes at room temperature and permeabilized (0.5% bovine serum albumin, 0.1% Triton (VWR International, Radnor, PA), and 2 mmol/L EDTA in phosphate-buffered saline) for 30 minutes at room temperature before blocking and staining according to the earlier-described protocols.