Ala amc

Purified recombinant human enzymes were purchased from R&D Systems (Minneapolis, MN). The quenched peptide substrate for aminopeptidase N was Ala-AMC (Bachem 1410, Torrance, CA). The substrate for puromycin-sensitive aminopeptidase was Leu-AMC (Bachem 1240). The substrate for all remaining enzymes was MCA-RPPGFSAFK-Dnp-OH (R&D Systems). Each substrate was diluted to 100 μM in the assay buffer recommended for each enzyme by the supplier. To a black microtiter plate (Corning 3686), 5 μl of compound dilutions in assay buffer were aliquoted in triplicate. To these, 5 μl of diluted enzyme was added, followed by 10 μl of the quenched peptide substrate. The plates were incubated at 37°C until at least 10% substrate turnover or 5 μM product had formed. Fluorescence was measured using a Tecan Safire II (Excitation 320 nm/Emission 420 nm). Each plate included a standard curve of reaction product and positive (no enzyme) and negative (dimethyl sulfoxide vehicle) controls. Dose-response data were plotted and inhibition values (IC₅₀) determined by linear regression analysis using GraphPad Prism (GraphPad Software, La Jolla, CA).

High glucose Dulbecco’s Modified Eagle’s Medium (DMEM), L-glutamine enriched Roswell Park Memorial Institute medium (RPMI) and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from Invitrogen. Hydroxylamine, glycine, sodium hydroxide, dibasic sodium phosphate and dimethyl sulfoxide (DMSO) were obtained from Sigma. Acetonitrile was obtained from Fisher. Hydrochloric acid, trifluoroacetic acid (TFA) mass spectroscopy grade and C-18 spin columns were purchased from Pierce Thermo Scientific. MG132, MG262 and clasto-Lactacystin β-lactone were purchased from Boston Biochem. AM114, butabindide and puromycin were purchased from Tocris Bioscience. Other inhibitors and their commercial sources were bortezomib (LC Laboratories), MLN2238 (Selleckchem), carfilzomib (ChemieTek), bestatin (Sigma), and bestatin methylester (Calbiochem). Recombinant human puromycin-sensitive aminopeptidase was purchased from R&D Systems. Suc-Leu-Leu-Val-Tyr-AMC, Ala-Ala-Phe-AMC, Leu-AMC and Ala-AMC were procured from Bachem. The isotopic labeling reagents 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) containing either 0, 3, 6, or 9 atoms of deuterium (D0-, D3-, D6-, and D9-TMAB-NHS, respectively) or 9 atoms of deuterium and three ¹³C atoms (D12-TMAB-NHS) were synthesized as described [30] .

To evaluate the expression of APN on the cell surface, HCC cells and normal hunman liver cell line THLE-3 were incubated with APC-conjugated anti-human APN/CD13 (Biolegend, San Diego, CA, USA) or APC Mouse IgG1, κ Isotype Ctrl (Biolegend) antibodies for 30 min at 4 °C. Then, the cells were analyzed by using a FACS Canto^TM II flow cytometer (BD Biosciences, San Jose, CA, USA).
APN activities in different cell lines were estimated by measuring the hydrolysis of a fluorescent substrate derived from alanine conjugated to 7-amido-4-methylcoumarin (Ala-AMC, Bachem, Budendorf, Switzerland), as described previously^{19 (link)}. The THLE-3 cell line was used as control.