prommp 9  (Cell Signaling Technology Inc)

Journal: Reproductive Biology and Endocrinology

Article Title: Spatio-temporal expression of MMP-2, MMP-9 and tissue kallikrein in uteroplacental units of the pregnant guinea-pig (Cavia porcellus)

doi: 10.1186/1477-7827-5-27

Figure Lengend Snippet: Western blot analysis of guinea-pig placental homogenates for MMP-9. A , Representative blot at days 20, 40 and 60 of pregnancy. Purified human proMMP-9 (hProMMP-9) yielded two bands with approximate molecular weights of 92 and 68 kDa. B , Mean ratio of active/total MMP-9 in five placental homogenates for each period. **P < 0.01 for 20 vs . 40 days; ***P < 0.001 for 40 vs . 60 days. DSCF posthoc comparisons after Kruskal-Wallis test.

Article Snippet: Standards were purified human recombinant proMMP-2, proMMP-9 (20 ng, Calbiochem, La Jolla, CA), and purified rat urinary kallikrein (5 ng) [ ].

Techniques: Western Blot, Purification

Journal: Reproductive Biology and Endocrinology

Article Title: Spatio-temporal expression of MMP-2, MMP-9 and tissue kallikrein in uteroplacental units of the pregnant guinea-pig (Cavia porcellus)

doi: 10.1186/1477-7827-5-27

Figure Lengend Snippet: Gelatin zymography of placental homogenates. A , Representative zymogram at days 20, 40 and 60 of pregnancy. Purified human proMMP-2 (hproMMP-2) and proMMP-9 (hproMMP-9) were used as standards. B-C , Densitometric analysis of zymograms from three placental homogenates at days 20, 40 and 60 for Pro/MMP-2 (B) and Pro/MMP-9 (C). Data is expressed with respect to the average densitometry at day 20. *, P < 0.05 for 40 vs . 20 and 60 days in (B); *, P < 0.05 for 40 and 60 vs . 20 days in (C). DSCF posthoc comparisons after Kruskal-Wallis test.

Article Snippet: Standards were purified human recombinant proMMP-2, proMMP-9 (20 ng, Calbiochem, La Jolla, CA), and purified rat urinary kallikrein (5 ng) [ ].

Techniques: Zymography, Purification

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: (A) Domain structure of MMP-9 with schematic representation of intact 92 kDa proMMP-9 and predicted fragment sizes by activation with MMP-3cd and APMA indicated. Purified TIMP-free MMP-9 (75 μg) was treated with either the MMP-3cd at 100 μM (B) or 2 mM APMA (C) for the indicated times. At each time point, 15 μg was removed for analysis by Coomassie protein staining or receptor blotting as indicated. Receptor binding was detected using monoclonal anti-LRP1 IgG 8G1 and goat anti-mouse IgG conjugated to HRP and visualized using chemiluminescence. MMP-9 is a 92 kDa zymogen. In the presence of either MMP-3cd or APMA, N-terminal cleavage of MMP-9 occurs yielding 86 and 82 kDa enzyme species (bracket). In the presence of APMA, the 82 kDa species undergoes C-terminal processing yielding a 68 kDa enzyme species (*).

Article Snippet: Anti-MMP-3 antibody (Cat#: MAB3306) and purified human proMMP-9 (Cat#: CC079, Uniprot entry P14780) were purchased from Chemicon.

Techniques: Activation Assay, Purification, Staining, Binding Assay

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Full length LRP1 was immobilized on a CM5 sensor chip using an amine-reactive coupling process. (A) proMMP-9 was injected over the chip in increasing concentrations of ligand (11.1, 33.3, 100, and 300 nM). (B) proMMP-9/TIMP-1 complexes were injected over the chip in increasing concentrations of ligand (3.7, 11.1, 33.3, 100, and 300 nM). Fits of the experimental data (black lines) to a bivalent binding model are shown as blue lines. The data shown is a representative experiment from two independent experiments that were performed.

Article Snippet: Anti-MMP-3 antibody (Cat#: MAB3306) and purified human proMMP-9 (Cat#: CC079, Uniprot entry P14780) were purchased from Chemicon.

Techniques: Injection, Binding Assay

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Cells were plated at 1 × 105 cells/well in a 12-well plate. A, B. 5 nM of 125I-labeled proMMP-9 (blue circles) or 125I-labeled proMMP-9/TIMP-1 (orange triangles) were incubated with mouse embryonic fibroblasts (MEFs) (A) or TIMP-1 KO MEFs (B) in the presence or absence of 1 μM RAP for indicated times and the amount internalized (left panel) or degraded (right panel) determined (n=2). C, D. 5 nM of 125I-labeled TIMP-1 (blue circles) or proMMP-9/125I-labeled TIMP-1 complex (orange triangles) were incubated with MEFs (C) or MMP-9 KO MEFs (D) in the presence or absence of 1 μM RAP for indicated times and the amount internalized (left panel) or degraded (right panel) determined (n=3). The data shown represent the RAP inhibitable fraction of internalized or degraded protein

Article Snippet: Anti-MMP-3 antibody (Cat#: MAB3306) and purified human proMMP-9 (Cat#: CC079, Uniprot entry P14780) were purchased from Chemicon.

Techniques: Labeling, Incubation

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

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: 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 < 0.001 when compared with PMNs incubated in the absence of exogenous Mmp. The results shown are representative of at least 3 independent experiments.

Article Snippet: The PMNs were then incubated at 4°C for 60 min with or without 500 nM purified murine hemopexin protein (Sino Biological, Wayne, PA), and then incubated at 4°C for 60 min with or without 400 nM proMmp-8 (R&D Systems, Minneapolis, MN) or 400 nM proMmp-9 (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Incubation, Software, Two Tailed Test

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

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Extracts of freshly-isolated unstimulated PMNs from WT and Timp-1−/− mice and cell-free supernatant fluids from WT and Timp-1−/− PMN that had been activated with 10−6 M fMLP for 30 min were immuno-blotted for Mmp-9 (A) or Mmp-8 (B). The arrows indicate the proMMP-9 and proMmp-8 forms of the Mmps. Note that extracts of unstimulated WT and Timp-1−/− PMNs contain similar amounts of proMmp-9 and proMmp-8 (Mr = 100 kDa for Mmp-9 and 85 kDa for Mmp-8). In addition, when activated with fMLP, Timp-1−/− PMNs freely release similar amounts of Mmp-8 and Mmp-9 exclusively as the latent proMmp forms.

Article Snippet: The PMNs were then incubated at 4°C for 60 min with or without 500 nM purified murine hemopexin protein (Sino Biological, Wayne, PA), and then incubated at 4°C for 60 min with or without 400 nM proMmp-8 (R&D Systems, Minneapolis, MN) or 400 nM proMmp-9 (R&D Systems, Minneapolis, MN).

Techniques: Isolation

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

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A, PAF- and fMLP-activated PMNs from Mmp-9−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-9, and then immunostained with rabbit anti-Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488. In B, PAF- and fMLP-activated PMNs from Mmp-8−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-8, and then immunostained with rabbit anti-Mmp-8 IgG, or non-immune rabbit IgG followed by goat- anti-rabbit F(ab)2-conjugated to Alexa 488. In A-B, the antibodies used detect both the pro and active forms of the Mmps. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with cells incubated without exogenous murine proMmp-9 or proMmp-8. In C, Timp-1−/− PMNs were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-9 for 1 h; 3) 200 nM Timp-1 for 1 h and then with proMmp-9 for 1 h; 4) 200 nM Timp-2 for 1 h, and then with proMmp-9 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in buffer. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-9. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In D, Timp-1−/− PMN were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-8 for 1 h; 3) 200 nM Timp-1 for 1 h and then proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then proMmp-8 for 1 h; 5) 200 nM myeloperoxidase for 1 h and then 200 nM proMmp-8 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in PBS. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-8. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In A-D, the results shown are representative of at least 3 independent experiments. In E-F, equal numbers of PAF- and fMLP-activated PMNs from Timp-1−/− mice were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and 200 nM proMmp-9 or proMmp-8 alone for 1 h; 3) 200 nM Timp-1 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; The cells were fixed and then incubated at 37°C with: 1) gelatin conjugated to quenched FITC for 18 h (in E); or 2) type-I collagen conjugated to quenched FITC for 18 h (in F). In E-F, Mmp-mediated cleavage of each substrate in the presence of amino-phenyl mercuric acetate was quantified in cell-free supernatant samples using fluorimetry, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate in cells incubated without exogenous proteins; n=4–5 separate experiments. Data were analyzed using a Kruskal-Wallis One-Way ANOVA followed by pair-wise testing with Mann-Whitney U tests. Asterisks indicate P < 0.05 compared with all other groups.

Article Snippet: The PMNs were then incubated at 4°C for 60 min with or without 500 nM purified murine hemopexin protein (Sino Biological, Wayne, PA), and then incubated at 4°C for 60 min with or without 400 nM proMmp-8 (R&D Systems, Minneapolis, MN) or 400 nM proMmp-9 (R&D Systems, Minneapolis, MN).

Techniques: Incubation, Two Tailed Test, MANN-WHITNEY

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

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A, PAF- and fMLP-activated Mmp-8−/− x Mmp-9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length proMmp-8 protein (FL Mmp8) or 400 nM mutant proMmp-8 protein lacking the COOH-terminal hemopexin domain (MT Mmp-8). Bound Mmp-8 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-8 (which is present in both the FL and MT forms of Mmp-8). In B, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length Mmp-9 (FL Mmp9) or 400 nM mutant Mmp-9 lacking the COOH-terminal hemopexin domain (MT Mmp9). Bound Mmp-9 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-9 (which is present in both the FL and MT forms of Mmp-8 and Mmp-9). In A-B, Data are mean + SEM; n = 3 separate experiments each analyzing 300 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 < 0.05 compared with the no exogenous Mmp group or the group indicated. In C-D, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated at 4°C for 60 min with or without 500 nM soluble murine hemopexin protein. Cells were then incubated at 4°C for 60 min with or without 400 nM exogenous proMmp-8 or 400 nM exogenous proMmp-9. Mmp-8 and Mmp-9 that bound to cells was detected by immunostaining the cells with Alexa-488 for Mmp-8 (C) or Mmp-9 (D), as described above. Data are mean + SEM; n = 3 separate experiments each analyzing 300 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 < 0.05 compared with the group incubated without exogenous proteins or the group indicated.

Article Snippet: The PMNs were then incubated at 4°C for 60 min with or without 500 nM purified murine hemopexin protein (Sino Biological, Wayne, PA), and then incubated at 4°C for 60 min with or without 400 nM proMmp-8 (R&D Systems, Minneapolis, MN) or 400 nM proMmp-9 (R&D Systems, Minneapolis, MN).

Techniques: Incubation, Mutagenesis, Immunostaining, Two Tailed Test