Journal: Journal of Clinical and Translational Hepatology

Article Title: Indole-3-propionic Acid-aggravated CCl 4 -induced Liver Fibrosis via the TGF-β1/Smads Signaling Pathway

doi: 10.14218/JCTH.2021.00032

Figure Lengend Snippet: (A) IHC staining of α-SMA, collagen-1, MMP-2, and TIMP-1 in the liver tissues (magnification 200×). (B–E) Effects of IPA on the mRNA expression of α-SMA, collagen-1, MMP-2, and TIMP-1 in the liver tissues measured by qPCR. All values were assessed by two independent experiments; the values represent the mean±SEM. n =3-4. * p <0.05, ** p <0.01, *** p <0.001. α-SMA, α-smooth muscle actin; HSCs, hepatic stellate cells; IHC, immunohistochemistry; IPA, indole-3-propionate; MMPs, matrix metalloproteinases; qPCR, quantitative real-time polymerase chain reaction; TIMPs, tissue inhibitors of metalloproteinase.

Article Snippet: Subsequently, sections were incubated overnight at 4°C with diluted primary antibodies, including α-SMA (1:2,000; GB13044; Servicebio, Inc. Wuhan, China), collagen I (1:1,200; GB11022-3; Servicebio, Inc.), matrix metalloproteinase (MMP)-2 (1:1,200; GB11130; Servicebio, Inc.), tissue inhibitors of metalloproteinase-1 (TIMP-1) (1:1,000; 106164-T40; Sino Biological US, Chesterbrook, PA, USA), TGF-β1 (1:500; GB11179; Servicebio, Inc.), and incubated with a conjugated secondary antibody after washing with phosphate-buffered saline (G0002; Servicebio, Inc.).

Techniques: Immunohistochemistry, Expressing, Real-time Polymerase Chain Reaction

Journal:

Article Title: Acquisition of Plasmin Activity by Fusobacterium nucleatum subsp. nucleatum and Potential Contribution to Tissue Destruction during Periodontitis

doi:

Figure Lengend Snippet: Degradation of TIMP-1 by u-PA-activated plasminogen-coated F. nucleatum subsp. nucleatum. Bacteria were incubated with TIMP-1 for 16 h at 37°C, and the supernatants were analyzed by SDS-PAGE and Western immunoblotting using an anti-TIMP-1 antibody. Lanes: 1, molecular mass markers (kDa); 2, TIMP-1 alone; 3, u-PA-activated plasminogen-coated bacteria with TIMP-1; 4, pure plasmin with TIMP-1; and 5, plasminogen-coated bacteria with TIMP-1.

Article Snippet: Thereafter, the membrane was incubated for 2.5 h with rabbit anti-native human TIMP-1 (Cedarlane Laboratories Ltd.) as the primary antibody at a concentration of 4 μg/ml, and then with goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate (Bio-Rad Laboratories, Mississauga, Ontario, Canada) as the secondary antibody at a dilution of 1/3,000 for 60 min.

Techniques: Incubation, SDS Page, Western Blot

Journal: Developmental neuroscience

Article Title: Ischemic-Reperfusion Injury Increases Matrix Metalloproteinases and Tissue Metalloproteinase Inhibitors in Fetal Sheep Brain

doi: 10.1159/000489700

Figure Lengend Snippet: Representative Western immunoblots and bar graphs of TIMP-1 (a), TIMP-2 (b), TIMP-3 (c), and TIMP-4 (d) expression in the fetal cerebral cortex obtained from the sham (n = 5), I/R-4 (n = 5), I/R-24 (n = 5), and I/R-48 (n = 5) sheep. TIMP-1, −2, and −3 expression levels were higher 24 than 4 h after ischemia. TIMP-1 and −3 expression levels were also higher in the I/R-24 group than in the sham group. TIMP-4 did not differ among groups (KruskalWallis, p = 0.15). Means ± SD. IC, internal control. * p < 0.05 vs. sham, + p < 0.05 vs. I/R-4 (ANOVA).

Article Snippet: We probed the immunoblotted membranes with primary rabbit polyclonal antibodies for MMP-8 and MMP-13 (Bioss Antibodies, Woburn, MA, USA), TIMP-1 (EMD Millipore, Billerica, MA, USA), TIMP-2, and TIMP-4 (Abbiotec LLC, San Diego, CA, USA) at a dilution of 1:5,000 and used primary mouse monoclonal antibodies to probe for TIMP-3 (Lifespan Biosciences, Seattle, WA, USA) at a dilution of 1:5,000, and for MMP-2 and MMP-9 (biotin-conjugated; Lifespan Biosciences), vinculin (Thermo Fisher Scientific-), and β-actin (Bio-Rad Laboratories) at a dilution of 1:10,000.

Techniques: Western Blot, Expressing

Journal: Developmental neuroscience

Article Title: Ischemic-Reperfusion Injury Increases Matrix Metalloproteinases and Tissue Metalloproteinase Inhibitors in Fetal Sheep Brain

doi: 10.1159/000489700

Figure Lengend Snippet: TIMP-1, −2, and −3 protein expression as the ratio to the internal control (IC) standard plotted against MMP-8 and −13 values normalized as the ratio to IC. Linear regression analysis shows a positive correlation between TIMP-1 and MMP-8 (r = 0.67, n = 17, p < 0.01), between TIMP-2 and MMP-8 (r = 0.71, n = 20, p < 0.001), and between TIMP-3 and MMP-8 (r = 0.77, n = 19, p < 0.001), between TIMP-1 and MMP-13 (r = 0.66, n = 17, p < 0.01), between TIMP-2 and MMP-13 (r = 0.76, n = 20, p < 0.001), and between TIMP-3 and MMP-13 (r = 0.74, n = 19, p < 0.001). Regression lines (black line) and 95% confidence bands (dashed lines) are shown.

Article Snippet: We probed the immunoblotted membranes with primary rabbit polyclonal antibodies for MMP-8 and MMP-13 (Bioss Antibodies, Woburn, MA, USA), TIMP-1 (EMD Millipore, Billerica, MA, USA), TIMP-2, and TIMP-4 (Abbiotec LLC, San Diego, CA, USA) at a dilution of 1:5,000 and used primary mouse monoclonal antibodies to probe for TIMP-3 (Lifespan Biosciences, Seattle, WA, USA) at a dilution of 1:5,000, and for MMP-2 and MMP-9 (biotin-conjugated; Lifespan Biosciences), vinculin (Thermo Fisher Scientific-), and β-actin (Bio-Rad Laboratories) at a dilution of 1:10,000.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase-14 Both Sheds Cell Surface Neuronal Glial Antigen 2 (NG2) Proteoglycan on Macrophages and Governs the Response to Peripheral Nerve Injury *

doi: 10.1074/jbc.M114.603431

Figure Lengend Snippet: The MMP-14/MMP-2/TIMP-2 axis in the PNS. A, immunostaining for MMP-14 using 3G4 antibody (2′2-diaminobenzidine; brown) in rat sciatic nerves at day 0 (normal) and days 1, 3, and 7 after crush injury (the crush site). MMP-14 is observed in Schwann cells (crescent-shaped; insets) and vessel (V) endothelial cells in all nerves and in macrophage-like cells at day 3 postinjury (asterisk). Images are representative of n = 3–4/group. Scale bars are 25 μm. B, TaqMan qPCR of MMP-14, MMP-2, and TIMP-2 in rat sciatic nerves at day 0 (normal (N)) and days 1, 3, and 7 after crush injury. The mean relative mRNA of n = 4/group are normalized to GAPDH and compared with the normal nerve samples (p values by analysis of variance and Bonferroni post hoc test) is shown. C, immunoblotting for MMP-14 (60 kDa), MMP-2 (72 and 68 kDa, latent and active, respectively), and TIMP-2 (21 kDa) in rat sciatic nerve at day 0 (normal) and days 1, 3, and 7 after crush injury. The graph represents the mean optical density of n = 4/group as a percentage of β-actin (p values by analysis of variance and Bonferroni post hoc test). D, immunostaining for MMP-14 (3G4 antibody; green) in the injured nerve with Schwann cells (S100; top, red) or macrophages (Iba1; bottom, red) depicts co-localization of the signals in the injured nerve (arrowhead). E, immunostaining of MMP-14 (3G4 antibody; green) and TIMP-2 (red) in rat sciatic nerve at day 3 postcrush. Schwann cells co-express MMP-14 and TIMP-2 (crescent-shaped structures; white arrows). TIMP-2−/MMP-14+ structures are observed (yellow arrowhead). D and E, all sections show DAPI-stained nuclei (blue) and vessels (V). Images are representative of n = 3/group. Scale bars are 25 μm. Error bars represent S.E.

Article Snippet: The following antibodies were also used in our experiments: rabbit polyclonal S100 antibody (Z0311, Dako), murine monoclonal CD68 antibody (MCA341R, Serotec), rabbit polyclonal Iba1 antibody (019-19741, Wako), rabbit polyclonal laminin antibody (L9393, Sigma), murine monoclonal β-actin antibody (A53166, Sigma), and rabbit polyclonal TIMP-2 antibody (C0348, Assay Biotechnology).

Techniques: Immunostaining, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase-14 Both Sheds Cell Surface Neuronal Glial Antigen 2 (NG2) Proteoglycan on Macrophages and Governs the Response to Peripheral Nerve Injury *

doi: 10.1074/jbc.M114.603431

Figure Lengend Snippet: The MMP-14/MMP-2/TIMP-2 axis in Schwann cells in vitro. A, TaqMan qPCR amplification plots for MMP-14, TIMP-2, and GAPDH (normalizer) in primary rat Schwann cell cultures (grown in DMEM containing 10% FBS for 24 h) and normal rat sciatic nerve. MMP-14 and TIMP-2 amplification closely follows GAPDH amplification (i.e. 0–5 cycle intervals between threshold cycle (Ct) values), suggesting high baseline expression of both the enzyme and its inhibitor. Data shown are from duplicate Schwann cell samples (same color curves) from two independent experiments or duplicate nerve samples (same color curves) pooled from n = 5/sample. B, immunoblotting for MMP-14 (AB8345 antibody) and TIMP-2 of Schwann whole cell lysate aliquots (5 μg/lane) and gelatin zymography of the Schwann cell medium aliquots (20 μl) for the activation status of MMP-2. β-Actin is used as a loading control. dRn, the fluorescence emission of the baseline. C, immunoblotting of MMP-14 (AB8345 antibody) in MCF7-mock, MCF7-MMP14, and Schwann cell whole cell lysate aliquots (equal amounts; 3.5 μg/lane each). D, imaging of the catalytically active cellular MMP-14. MCF7-mock, MCF7-MMP14, and Schwann cells were co-incubated for 3 h with the MP-3653 fluorescent reporter alone or jointly with the non-fluorescent hydroxamate inhibitor GM6001 (+GM6001). The resulting fluorescence of the cell-bound MP-3653 reporter recorded active MMP-14 (green). DAPI stains the nuclei (blue). Scale bars are 8 μm. E, Schwann cells immunostaining with the MMP-14 3G4 antibody are reactive with both active and inactive enzyme (green). Scale bars are 15 μm.

Article Snippet: The following antibodies were also used in our experiments: rabbit polyclonal S100 antibody (Z0311, Dako), murine monoclonal CD68 antibody (MCA341R, Serotec), rabbit polyclonal Iba1 antibody (019-19741, Wako), rabbit polyclonal laminin antibody (L9393, Sigma), murine monoclonal β-actin antibody (A53166, Sigma), and rabbit polyclonal TIMP-2 antibody (C0348, Assay Biotechnology).

Techniques: In Vitro, Amplification, Expressing, Western Blot, Zymography, Activation Assay, Fluorescence, Imaging, Incubation, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase-14 Both Sheds Cell Surface Neuronal Glial Antigen 2 (NG2) Proteoglycan on Macrophages and Governs the Response to Peripheral Nerve Injury *

doi: 10.1074/jbc.M114.603431

Figure Lengend Snippet: NG2-Mφ in the PNS and in cultures stimulated with MMP-14. A, immunostaining of NG2 (green) and CD68 (red) in rat sciatic nerve at day 0 (normal) and days 3 and 7 after crush injury. NG2+/CD68+ cells (white arrowheads) and NG2+/CD68− cells adjacent to NG2−/CD68+ Mφ (yellow arrows) are observed. Scale bars are 25 μm. B, immunostaining of MMP-14 (3G4 antibody; green) and NG2 (AB5320 antibody; red) in sciatic nerve at days 3 and 7 postcrush. White arrowheads, NG2 co-localized with MMP-14 in fibroblast-like cells and microvascular (V) pericytes and/or endothelial cells especially at day 7 postcrush. Yellow arrows, MMP-14 and NG2 interface when expressed by adjacent cells. Scale bars are 30 μm. C, immunostaining of Iba1 (red) and CD68 (green) in rat sciatic nerve at day 7 postcrush. Macrophages recruited into the injury site are predominantly CD68+ phagocytes. Scale bars are 20 μm. D, immunostaining of NG2 (AB5320 antibody; green) and CD68 (red) in the TBI lesion epicenter shows NG2+/CD68+ cells (white arrowheads). Scale bar are 20 μm. A–D, images are representative of n = 4/group. E, immunostaining of NG2 (AB5320 antibody; green) and CD68 (red) in the fixed (4% PFA) NG2-Mφ cultures isolated from the TBI lesion epicenter from D. Images are representative of brain tissues from about n = 5/group. Scale bars are 25 μm. A–E, DAPI stains the nuclei (blue). F, immunoblotting of NG2 (05-710 antibody) in the cultured NG2-Mφ lysates from E. Where indicated, the cells were incubated with MMP-14 (1 μg/ml) alone or jointly with TIMP-2 (50 ng) or GM6001 (10 μm) for 30 min. Data are representative of two independent experiments with brain tissues from about n = 5/group. G, immunoblotting of NG2 (05-710 antibody) in sciatic nerve at days 0 (normal) and 7 postcrush. Duplicate representative samples of n = 4/group are shown. F and G, β-actin is used as a loading control.

Article Snippet: The following antibodies were also used in our experiments: rabbit polyclonal S100 antibody (Z0311, Dako), murine monoclonal CD68 antibody (MCA341R, Serotec), rabbit polyclonal Iba1 antibody (019-19741, Wako), rabbit polyclonal laminin antibody (L9393, Sigma), murine monoclonal β-actin antibody (A53166, Sigma), and rabbit polyclonal TIMP-2 antibody (C0348, Assay Biotechnology).

Techniques: Immunostaining, Isolation, Western Blot, Cell Culture, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Matrix Metalloproteinase-14 Both Sheds Cell Surface Neuronal Glial Antigen 2 (NG2) Proteoglycan on Macrophages and Governs the Response to Peripheral Nerve Injury *

doi: 10.1074/jbc.M114.603431

Figure Lengend Snippet: MMP-14 translocation toward the node of Ranvier (NR). Immunostaining of MMP-14 (3G4 antibody; green) and NG2 (AB5320 antibody; red), TIMP-2 (red), or laminin (red) in teased out myelinated nerve fibers in rat sciatic nerves at days 0 (normal; A) and 3 after transection (B) is shown. Control (C) is stained using species-specific secondary antibodies conjugated to Alexa Fluor 488 (green) and Alexa Fluor 594 (red). White arrows indicate MMP-14 co-localized with its substrates, NG2 and laminin, postinjury. White arrowheads indicate TIMP-2−/MMP-14+ reactivity. Yellow arrows indicate intraaxonal TIMP-2 staining. Images are representative of ∼40/group. Scale bars are 5 μm.

Article Snippet: The following antibodies were also used in our experiments: rabbit polyclonal S100 antibody (Z0311, Dako), murine monoclonal CD68 antibody (MCA341R, Serotec), rabbit polyclonal Iba1 antibody (019-19741, Wako), rabbit polyclonal laminin antibody (L9393, Sigma), murine monoclonal β-actin antibody (A53166, Sigma), and rabbit polyclonal TIMP-2 antibody (C0348, Assay Biotechnology).

Techniques: Translocation Assay, Immunostaining, Staining