Journal: Journal of immunology (Baltimore, Md. : 1950)
Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces
Figure Lengend Snippet: ProMMP-8 and proMMP-9 share binding sites on the surface of PMNs: In A and B , PMNs from Mmp-8 −/− x Mmp-9 −/− mice were activated for 15 min at 37°C with 10 −6 M PAF followed by 10 −6 M fMLP to induce surface expression of Timp-1. In A , the activated PMNs were then pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-8 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous murine proMMP-9. Cells were then washed and immunostained with rabbit anti-murine Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab) 2 -conjugated to Alexa 488 to detect bound pro and active Mmp-9. In B , the PAF- and fMLP- activated and PMNs were pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-9 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous full length murine proMMP-8. Cells were then immunostained with rabbit anti-murine Mmp-8 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab) 2 -conjugated to Alexa 488 to detect bound pro and active Mmp-8. In A and B , images of immunostained cells were captured and surface-bound pro and active Mmp-9 (in A ) or pro and active Mmp-8 (in B ) were quantified using MetaMorph software. Data are mean + SEM (n = 150–200 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P
Article Snippet: Briefly, samples were subjected to electrophoresis on 8% SDS-PAGE under reducing conditions, proteins transferred to PVDF membranes, and probed with polyclonal rabbit anti-murine Mmp-9 IgG (AB19047) or rabbit anti-murine Mmp-8 IgG (Ab19045), followed by goat anti-rabbit IgG conjugated to horse radish peroxidase (Biorad, Hercules, CA), and a chemiluminescence detection system following the manufacturer’s instructions.
Techniques: Binding Assay, Mouse Assay, Expressing, Incubation, Software, Two Tailed Test