Comprehensive Enzymatic Activity Profiling

MMP and cathepsin gene expression was performed by quantitative PCR (qPCR) using validated Taqman probes (Life Technologies/Applied Biosystems, Carlsbad, CA). All qPCR results are represented as relative quantification (RQ) compared to the mock treated animals and corrected to actin levels. Several MMP levels were measured in BALF and cell culture supernatants using a bead assay (MILLIPLEX MAP MMP Magnetic Bead Panels, EMD Millipore, Billerica, MA) with the BioRad Bio-Plex 200 system (BioRad, Hercules, CA). Cathepsin tissue levels were determined by immunoblots and BALF cathepsin S and B activity assays as previously described^{46 (link)}. Cathepsin G BALF activity was determined by a commercial available colorimetric assay (Abcam; ab126780). BALF collagenase activity was determined by a colorimetric ninhydrin method, as previously described^{22 (link)}. Proteinase activity was also assessed with the same colorimetric ninhydrin method but utilizing casein as the substrate. BALF elastase activity was measured by the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide(Suc Ala₃NA) per minute at 25°C and pH 8.0. BALF gelatinase activity was determined by gelatin zymography.