Bestatin

APN enzymatic activity was determined by colorimetric measurement of the hydrolysis of the substrate L-alanine 4-nitroanilide hydrochloride (Sigma, USA), as described previously [17 (link)]. Briefly, 1 × 10⁵ cells (in 50 µl PBS with 10% FBS) were placed in flat-bottom wells of 96-well culture plates and were preincubated with the indicated mAb or Bestatin for 1 h at 37°C. Bestatin (Santa Cruz Biotechnology, Dallas TX, USA), a well-known CD13 inhibitor, was added at the maximal inhibitory doses of 4 µg/ml for U-937 cells and 40 µg/ml for HEK-ANPEP. After incubation of the cells with the mAbs or with Bestatin, substrate was added to a final concentration of 6 mM and the plates were incubated for 1 h at 37°C. Cells were pelleted, and the absorbance of the supernatants at 405 nm was determined immediately. Determinations were performed in triplicate wells. Data are presented as percentage of the activity of control cells, which were not incubated with mAbs or Bestatin before addition of the substrate.

Histopaque, p-nitrophenyl valerate (pNPVa), and LPS (E. coli 055:B5) were purchased from Sigma (St. Louis, MO, USA). AIM V^® Serum Free Medium was purchased from Thermo-Fisher (Waltham, MA, USA). RIPA lysis buffer and protease inhibitors (phenylmethylsulfonyl fluoride, PMSF; 4-(2-aminoethyl)benzenesulfonyl fluoride, AEBSF; bestatin; pepstatin A; leupeptin hemisulfate; and aprotinin) were from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies used for Western blots (anti-CES1, anti-MAGL, β-actin, goat anti-rabbit, and goat anti-mouse) were purchased from Abcam (Cambridge, MA, USA). Authentic 2-AG, anandamide, AA, and its deuterated analog AA-d8 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Small molecules JZL184 and WWL113 were purchased from Sigma. CpG was purchased from InvivoGen (San Diego, CA, USA). Primary CES1 and MAGL antibodies for flow cytometry were purchased from Abcam. Fluorescence secondary antibodies for flow cytometry and antibodies against IL-6 used to neutralize IL-6 or perform ELISA were purchased from Biolegend (San Diego, CA, USA). For some of the experiments, monocyte-depleted (n = 1) and whole PBMCs (n = 5) were purchased from Astarte Biologics (Bothell, WA, USA).