Arg arg β naphthylamide

Total protein extracts were prepared from tail biopsies minced in 150 μL Tris–HCl, pH 7.8, and subjected to sonication and freeze-thawing cycles. For protein extracts from total bone, the tissue was frozen in liquid nitrogen and homogenized using aluminum beads and the TissueLyser II instrument (Qiagen, Hilden, Germany). Tissue debris were removed by centrifugation at 4°C. Protein concentration in the supernatants were estimated using the DC™ Protein Assay Kit II (Bio-Rad, Berkeley, CA, USA) following the manufacturer’s instructions, on a Synergy™ H4 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). DPP3 enzymatic activity was performed as reported,^{(31 (link))} with minor modifications. Briefly, 50 to 80 μg of protein extracts were assayed with 0.04 mM Arg-Arg-β-naphthylamide (Sigma-Aldrich, Saint Louis, MO, USA) in 250 μL Tris–HCl, pH 8.5, at room temperature (RT). The reaction was stopped by adding 50 μL of 2 M acetate buffer, pH 4.5, containing 10% Tween and 1.5 mg/mL Fast Blue B Salt (all chemicals from Sigma-Aldrich). The absorbance of the released product (β-naphthylamine) was measured at 530 nm, using SynergyTM H4 Microplate Reader. The enzymatic activity was expressed as percentage of the wild-type (WT) value.