Arginase1

Immunohistochemistry analysis was performed in five µm-thick frozen or formalin-fixed paraffin-embedded liver tissue sections using antibodies against CYP3A (Biotrend, Cologne, Germany), CYP1A, CYP2C (a gift from Dr. R. Wolf, Biochemical Research Centre, University of Dundee, Dundee, UK), CYP2E1, Arginase1 (Sigma-Aldrich Corp., St. Louis, MO, USA), GS (BD Bioscience, Heidelberg, Germany), and CPS1 (Abcam, Cambridge, UK) (Table 1). The following horseradish peroxidase-conjugated secondary antibodies were used: anti-rabbit IgG (Agilent, Santa Clara, CA, USA), anti-mouse IgG (Sigma-Aldrich Corp., St. Louis, MO, USA), and anti-rat IgG (Linaris GmbH, Heidelberg, Germany) (Table 1). In order to visualize the target signal, the tissues were stained with either 3,3′-diaminobenzidine solution (Vector Laboratories, Peterborough, UK) or AEC+ high sensitivity substrate chromogen (Agilent, Santa Clara, CA, USA). The nuclei were visualized by counter-staining with Mayer’s haematoxylin.

Primary antibodies were purchased from the following sources and utilized at the following dilutions: VE-cadherin (F-8, Santa Cruz Biotechnology; 1:200), Arginase-1 (Sigma-Aldrich; 1:400), acetylated α-tubulin (6-11B-1, Santa Cruz Biotechnology; 1:100), and HNF4α (C-19, Santa Cruz Biotechnology; 1:400). Dylight 649-conjugated Ulex Europaeus Agglutinin I lectin (1:200) was purchased from Vector Laboratories. For secondary antibodies, Alexa Fluor 488, 568, 594, and 647 anti-mouse, anti-goat, and anti-rabbit IgG secondary antibodies were purchased from Life Technologies.

Total protein lysates were prepared with NP40 lysis buffer (Boston BioProducts) with Protease inhibitors (Complete, Roche). Proteins were quantitated and 50 μg protein was resolved on SDS gel. Proteins were then transferred to PVDF membrane overnight in cold room. Blot was incubated with blocking buffer (5% milk in 1X TBST) for one hour at room temperature and then with Arginase 1 (Sigma, SAB2108087) and tubulin antibodies (Cell Signaling, #2148) overnight in cold room on a shaking platform. After washing 3 times with 1X TBST (each 10 min at room temperature), HRP conjugated secondary antibody was added to the blot for another hour at room temperature. The blot was again washed 3 times with 1X TBST, then ECL reagent was used to reveal the signals on Fluro Chem (Protein Simple). Arg1 mRNA expression information was obtained from the Cancer Cell line Encyclopedia (CCLE) [41 (link)].