Mab936

According to the Laemmli method, all samples (10 μg of protein) were electrophoresed on SDS-polyacrylamide gel (10%) [29 (link)], blotted to nitrocellulose membranes (Sigma-Aldrich; Saint Louis, MO, USA) at 100 mA for 1 h. The membranes were blocked using 5% (w/v) nonfat powdered milk in the solution of TBS-T (20 mM Tris/HCl buffer, pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20) for 1 h. Then, samples were incubated overnight at 4 °C with antibodies against metalloproteinase-2 (Cat#MAB9021; R&D Systems, USA) or, respectively, directed against metalloproteinase-9 (Cat# MAB936; R&D Systems; USA) in TBS-T which contained 1% bovine serum albumin (w/v). In the next step, several washes in TBS-T buffer were performed. Bounded antibodies were detected using alkaline phosphatase (ALP) coupled with the appropriate antibody in the same solution at room temperature for 1 h with moderate shaking, and then BCIP/NBT reagent was used (Cat# B1911; Sigma; USA). Pre-stained molecular mass markers were used to determine the molecular mass of matrix metalloproteinases (BioRad, Hercules, CA, USA). Representative blots were demonstrated.

Rabbits were immunized with the peptide (NH₂)-FQTFEGDC conjugated to both ovalbumin and keyhole limpet hemocyanin. Based on serum titers toward the immunizing peptide, animals were selected for hybridoma library generation (Abcam, Burlingame, CA, USA). In total, 40,000 individual hybridomas were screened for immunoreactivity of supernatants with the immunizing peptide. Positive supernatants were screened by Western blot with full-length pro- and active-MMP9 recombinant standards [28 (link)]. Positive hybridoma supernatants were screened by IHC against a series of formalin-fixed HEK293 cell pellets containing active MMP9 (see Appendix A for details). Immunoglobulin G complementary DNA (cDNA) was sequenced from the positive hybridomas by 5′RACE (Abcam), cloned into pcDNA3.1 expression vector (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), expressed in HEK293 cells, and purified by protein-A chromatography (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). Sandwich immunoassays were conducted by coating the plate in 1 μg/mL recombinant antibody, adding recombinant MMP9 standard ADX, detecting with total MMP9 antibody (MAB936; R&D Systems, Minneapolis, MN, USA), and quantifying with a ruthenium-conjugated anti-mouse antibody (Meso Scale Discovery, Rockville, MD, USA).

Following isolation, neutrophils were pelleted, and the cell pellets were lysed using RIPA lysis buffer supplemented with a protease inhibitor cocktail. After centrifugation (12,000× g), the proteins were quantified by bicinchoninic acid (BCA) Protein Assay Kit. Samples of 30 proteins were separated on 10% or 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis and transferred to nitrocellulose membranes. The resulting blots were subsequently probed with specific primary antibodies directed against IL-1β (MAB601, R&D Systems), S100A9 (PA1-46489, ThermoFisher, Waltham, MA, USA), MCP-1 (ab9669, Abcam), MyD88 (AF2928, R&D Systems), p22phox (SC-271262, Santa-Cruz Biochtenology, Dallas, TX, USA), MMP-9 (MAB936, R&D Systems), Neutrophil Elastase (MAB91671-100, R&D Systems), MPO (AF3667, R&D Systems), GAPDH (ab9485, Abcam, Cambridge, UK), and β-tubulin (ab6046, Abcam). These were followed by secondary antibodies: anti-rabbit HRP-coupled (HAF008, R&D Systems), anti-mouse HRP-coupled (#31430, R&D Systems), or anti-goat HRP-coupled (HAF017, R&D Systems). The signals were visualized using SuperSignal West Pico chemiluminescent substrate (Pierce, Appleton, WI, USA) and quantified by densitometry employing a gel analyzer system, a LAS 4000 luminescent image analyzer (Fujifilm, Minato City, Tokyo, Japan), and image reader LAS 4000 software.