Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: Key resources

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques: Recombinant, Diagnostic Assay, Plasmid Preparation, Staining, Extraction, Sterility, Saline, Construct, Electron Microscopy, Expressing, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Multiplex Assay, Transfection, Sequencing, Software, Control

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: Characterization of TIMP2 protein constructs

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques: Concentration Assay

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: Description of mouse cohorts

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques:

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: Hippocampal gene expression following treatment with TIMP2 and TIMP2-hIgG4

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques: Gene Expression

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: Few significant changes in hippocampal gene expression following treatment with TIMP2 or TIMP2-hIgG4

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques: Gene Expression

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: The TIMP2 constructs had distinct MMP inhibitory profiles

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques: Construct, Inhibition

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: The alanine insertion into TIMP2 prevented MMP inhibitory activity at biologically relevant concentrations

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques: Activity Assay

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: MMP protein constructs. Schematic depicting the nine MMP protein constructs assessed for binding to TIMP2 constructs using bio-layer interferometry (BLI) and surface plasmon resonance (SPR). CD, catalytic domain.

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques: Construct, Binding Assay, SPR Assay

Journal: eNeuro

Article Title: Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging

doi: 10.1523/ENEURO.0031-23.2023

Figure Lengend Snippet: Characterization of TIMP2-MMP binding of the TIMP2 constructs

Article Snippet: Response 3: To measure mouse TIMP2, we used an ELISA from R&D Systems (Cat #: DY6304; https://www.rndsystems.com/products/mouse-timp-2-duoset-elisa_dy6304-05).

Techniques: Binding Assay

Journal: Cell and Tissue Research

Article Title: MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

doi: 10.1007/s00441-012-1429-1

Figure Lengend Snippet: a Gelatin zymograms of detergent extracts ( RIPA ) of SSpH reveal MMP and serine proteinases as components of the IAM. Blockage of the 72-kDa enzymatic activity with GM6001 (Ilomastat) ( lane 2 ) indicates a metalloprotease and blockage of the 35-kDa enzymatic activities by trypsin inhibitor ( lane 3 ) indicates serine proteases. Lane 4 , a positive control, is loaded with a trophoblast cell medium containing both MMP2 (72 kDa) and MMP9 (92 kDa). b Gelatin zymogram without (control) and with (block) a cyclic disulfide bonded peptide (CTTHWGFTLC) on RIPA extracts of SSpH ( lane 1 ) and trophoblast media ( lane 2 ), containing both MMP2 and MMP9 enzymatic activities. c Immunoblotting verification that acrosin is responsible for serine protease activity in detergent extracts of SSpH. Anti-acrosin antibody detects proacrosin in western blots of whole bull epididymal sperm ( lane 2 ) and is blocked when preincubated with the peptide it was raised against ( lane 1 ). Sonication of whole sperm causes proacrosin cleavage into its active forms as indicated both in the resultant sonication supernatant ( lane 3 ) and in SSpH ( lane 4 ). The 2 % NP-40 (non-ionic detergent) extract of SSpH ( lane 5 ) is less efficient in stripping SSpH of proacrosin/acrosin than 2 % SDS ( lane 4 ). Lane 6 is a gelatin zymogram of the NP-40 extract loaded in lane 5 , confirming that the enzymatic activities found at the 35-kDa level are due to acrosin. d Immunoblotting verification that both MMP2 and Proacrosin/Acrosin are constituents of the sonicated bull sperm head. Freeze–thawed sperm ( lane 1 ) were sonicated and separated by centrifugation into three fractions: supernatant ( lane 2 ), SSpH ( lane 3 ), and tails ( lane 4 ). The sperm heads were then extracted with SDS ( lane 5 ) and compared with pellet ( lane 6 ). The upper part of the western blot was probed with anti-pMMP2, while the bottom part below the demarcating line was probed with polyclonal anti-bull acrosin antibody. e Immunoblots probed with anti-tMMP2 showing that MMP2 is present in bull, mouse and human spermatozoa (WS). Human recombinant MMP2, minus the pre-domain, is used as a positive control (rec. MMP2)

Article Snippet: Oocytes were pre-incubated in HTF with or without anti-pMMP2 (1:50), anti-tMMP2 (1:25), pre-immune anti-tMMP2 IgG purified antibody (1:10) or 40nM tissue inhibitor of MMP2 (TIMP2; R&D Systems 971-TM, Minneapolis, MN, USA).

Techniques: Activity Assay, Positive Control, Control, Blocking Assay, Western Blot, Sonication, Stripping Membranes, Centrifugation, Recombinant

Journal: Cell and Tissue Research

Article Title: MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

doi: 10.1007/s00441-012-1429-1

Figure Lengend Snippet: Immunogold localization of MMP2 in bull spermatozoa and spermatid at end of cap phase. a In ejaculated sperm immunogold labeling, utilizing anti-tMMP2 antibody, is found through the apical ( AS ) and principal segments ( PS ) of the acrosome, a large portion associated with the IAM. See Suppl. Fig. b for preimmune control. Bar 0.2 μm. b In step 7-8 spermatid labeling with anti-pMMP antibody is seen along the inner acrosomal membrane ( arrows ) of the acrosome. AG acrosomic granule. Bar 0.2 μm

Article Snippet: Oocytes were pre-incubated in HTF with or without anti-pMMP2 (1:50), anti-tMMP2 (1:25), pre-immune anti-tMMP2 IgG purified antibody (1:10) or 40nM tissue inhibitor of MMP2 (TIMP2; R&D Systems 971-TM, Minneapolis, MN, USA).

Techniques: Labeling, Control, Membrane

Journal: Cell and Tissue Research

Article Title: MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

doi: 10.1007/s00441-012-1429-1

Figure Lengend Snippet: Immunoperoxidase staining of MMP2 (with anti-tMMP2 antibody) in a testicular section of round spermatids in step 2–3 of bovine spermiogenesis. a As with acrosin, MMP2 immunostaining is found in the acrosomic granule ( arrows ) of proacrosomic and acrosomic vesicles before shifting to the acrosomal membrane during the cap phase of spermiogenesis. b Preimmune control. Bars 5 μm

Article Snippet: Oocytes were pre-incubated in HTF with or without anti-pMMP2 (1:50), anti-tMMP2 (1:25), pre-immune anti-tMMP2 IgG purified antibody (1:10) or 40nM tissue inhibitor of MMP2 (TIMP2; R&D Systems 971-TM, Minneapolis, MN, USA).

Techniques: Immunoperoxidase Staining, Immunostaining, Membrane, Control

Journal: Cell and Tissue Research

Article Title: MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

doi: 10.1007/s00441-012-1429-1

Figure Lengend Snippet: Immunmoperoxidase staining of MMP2 in stages III and VII of the cycle of mouse seminiferous epithelium. a MMP2 immunostaining, utilizing anti-pMMP2 antibody, is associated with the acrosomic vesicles ( arrows ) of step 3 spermatids in stage III and the acrosomic cap of step 7 spermatids ( arrows ) in stage VII. The association of MMP2 with the acrosomic granule ( arrows ) of step 3 spermatids and the acrosomal membrane ( arrows ) of step 7 spermatids is seen to have a better advantage at higher magnification in the insets. Bars 10 μm. b Normal rabbit serum control

Article Snippet: Oocytes were pre-incubated in HTF with or without anti-pMMP2 (1:50), anti-tMMP2 (1:25), pre-immune anti-tMMP2 IgG purified antibody (1:10) or 40nM tissue inhibitor of MMP2 (TIMP2; R&D Systems 971-TM, Minneapolis, MN, USA).

Techniques: Staining, Immunostaining, Membrane, Control

Journal: Cell and Tissue Research

Article Title: MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

doi: 10.1007/s00441-012-1429-1

Figure Lengend Snippet: Effect of anti-MMP2 antibodies and TIMP2 in mouse IVF. IVF control values were adjusted to 100 % for comparative purposes and experimental values obtained were adjusted accordingly. The block graphs depict the mean percentage of oocytes that were fertilized after incubation with sperm in IVF medium containing anti-pMMP2, anti-SPACA1, anti-tMMP2, or TIMP2, as compared to controls. Superscripts a and b denote a significant difference at P < 0.05 and error bars indicate standard error. In ( a ), only the pre-incubation medium with spermatozoa contained antibodies of which only part was transferred to the final IVF medium. In ( b ) both spermatozoa and oocytes were preincubated with antibodies and inhibitors that were combined in the final IVF medium. Thus, it is difficult to distinguish if the difference in the effectiveness of inhibition in ( a ) and ( b ) is due to anti-MMP2 antibody effectiveness or concentration of antibody in IVF media

Article Snippet: Oocytes were pre-incubated in HTF with or without anti-pMMP2 (1:50), anti-tMMP2 (1:25), pre-immune anti-tMMP2 IgG purified antibody (1:10) or 40nM tissue inhibitor of MMP2 (TIMP2; R&D Systems 971-TM, Minneapolis, MN, USA).

Techniques: Control, Blocking Assay, Incubation, Inhibition, Concentration Assay

Journal: Journal of cell science

Article Title: MT1-MMP promotes vascular smooth muscle dedifferentiation through LRP1 processing.

doi: 10.1242/jcs.035279

Figure Lengend Snippet: Fig. 2. MT1-MMP promotes PDGFRβ- and PDGF-BB-dependent suppression of VSMC contractile proteins. (A) Quantitative assessment of SMA and calponin mRNA expression by real-time PCR. mRNA was isolated from subconfluent tenth-passage VSMCs cultured with the wide-spectrum MMP inhibitor BB-94 (10 μM), PDGFRβ kinase inhibitor AG1296 (10 μM) and EGFR tyrosine-kinase inhibitor AG1478 (10 μM) as indicated. The expression data are normalized against mouse TBP and presented relative to the mRNA expression in mock (0.1% DMSO)-treated wild-type cells (mean ±1 s.d., n=3). (B) Early-passage wild-type (MT1+/+) and MT1-MMP–/– VSMCs were cultured on polymerized collagen I in the presence of TIMP2 (4 μg/ml) for 48 hours as indicated, and exposed to PDGF-BB (25 ng/ml) under serum-free conditions. After 48 hours, the cells were lysed and the lysates analyzed by immunoblotting for the relative levels of calponin, SMA and vimentin. β-tubulin served as a loading control. Mean values of the ratios between normalized SMA and vimentin protein levels are presented below each lane (n=3). Relative mobilities of the molecular-mass markers are indicated in kDa. (C) Calponin expression was assessed by immunofluorescence staining of cells exposed to PDGF- BB as above. Filamentous actin was visualized with phalloidin (F-actin) and nuclei with DAPI. (D) MT1-MMP–/– VSMCs were transfected with EGFP expression-vector alone (Mock), or with either expression construct for wild-type MT1-MMP (MT1-MMP) or the catalytically inactive mutant (MT1- E240A) as indicated. Calponin expression (red) was assessed by immunofluorescence after 48 hours of treatment with PDGF-BB.

Article Snippet: In selected experiments, VSMCs were cultured or pre-treated and stimulated in the presence of human recombinant TIMP2 (4-5 μg/ml, R&D Systems), the synthetic MMP inhibitors BB-94 or GM6001 (5-10 μM final concentration in 0.1% DMSO), PDGFRβ kinase inhibitor (AG1296, 10 μm) and EGFR tyrosine-kinase inhibitor (AG1478, 10 μm; all from Calbiochem except BB-94 kindly provided by British Biotech).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Cell Culture, Western Blot, Control, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Construct, Mutagenesis