Z phe ala

Snap-frozen liver, spleen, kidney, and brain samples isolated from WT, untreated, and treated Ctsa^–/– mice from each group were homogenized on ice in three volumes of water (w/v) using the Qiagen TissueLyser II (Qiagen, Germantown, MD, USA). CA activity was measured against the dipeptide Z-Phe-Ala (Sigma, St. Louis, MO, USA), Neu1 catalytic activity was measured against synthetic 4-methylumbelliferyl-α-D-N-acetylneuraminic acid (Sigma, St. Louis, MO, USA), and β-GAL was measured against 4-methylumbelliferryl-β-D-galactopyranoside (Sigma, St. Louis, MO, USA). All reactions were performed at 37°C for 1 h and stopped with carbonate stop buffer (0.5 M Na₂CO₃ with the pH set to 10.7 by adding 0.5 M NaHCO₃) for Neu1 and β-GAL activities, while the CA activity was stopped at 100°C for 5 min, and 10 μL reaction mixture was read in 250 μL 50 mM sodium-carbonate stop buffer (pH 9.5), containing 500 μL o-phthaldialdehyde (10 mg/mL) and 500 μL 2-mercaptoethanol (5 μL/mL) per 30 mL. Fluorescence was measured at λ_ex 355/λ_em 460 and interpolated to a standard curve adjusted for dilution and normalized to protein concentration.