Sb 3ct

Bortezomib (sc-217786), azithromycin (sc-254949), minocycline (sc-203339) and SB-3CT (sc-205847) were purchased from Santa Cruz Biotechnology (TX, USA). LPS from Escherichia coli 0111:B4 (L4391) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human full-length proMMP-9 (92 kDa) was expressed in Sf9 insect cells and purified by gelatin-Sepharose chromatography. ProMMP-9 was activated by incubation with the catalytic domain of stromelysin-1/MMP-3 (cat. No. 444217, Merck Millipore, Darmstadt, Germany). The recombinant expression, purification and activation of proMMP-9 were performed as described previously [14 (link), 35 (link)].

All dairy goats were maintained according to the No. 5 proclamation of the Ministry of Agriculture, P.R. China. Sample collection was approved by the Institutional Animal Care and Use Ethics Committee of Northwest A&F University and performed in accordance with the ‘Guidelines for Experimental Animals’ of the Ministry of Science and Technology (Beijing, China). Three-year-old Xinong Saanen dairy goats at peak lactation (n = 6) slaughtered by captive bolt stunning followed by exsanguination [17 (link)]. Mammary gland, subcutaneous adipose, skeletal muscle, heart, liver, spleen, lung, kidney, rumen, marrow, and small intestine tissues were collected. All tissue samples were obtained under sterile conditions and washed with diethylpyrocarbonate (DEPC) treated water, then immediately frozen in liquid nitrogen. The Staphylococcus aureus strains were isolated from local Xinong Saanen goats and kept in our laboratory. SB-3CT, an inhibitor of MMP-9, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Neutrophil elastase activity was measured in the presence or absence of intact SLPI or SLPI treated with active MMP-9. For the gelatin degradation assay, NE was used at a final concentration of 5 nM and activity was monitored immediately after addition of 2.5 µg/ml DQ™-gelatin (Invitrogen, Carlsbad, CA, USA). To avoid background gelatinolysis by MMP-9, derived from the digestion of SLPI, all reactions were performed in the presence of 50 µM SB-3CT (Santa Cruz Biotechnology, Dallas, TX, USA). For a second activity test, we used the fluorogenic elastase substrate V (MeOSuc-Ala-Ala-Pro-Val-AMC) (Millipore, Burlington, MA, USA). 5 nM NE was combined with different concentrations and digestions of SLPI and incubated for 30 min at 37°C. Next, 20 µM elastase V substrate was added and fluorescence was measured every minute. Data derived from both experiments were fitted by linear regression and the velocity of the reaction was used as a measure for enzyme activity.