rabbit anti-mt3  (Millipore)

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus Induces TGF-β1 Activation in Renal Tubular Epithelial Cells after Epithelial-to-Mesenchymal Transition

doi: 10.1371/journal.ppat.1001170

Figure Lengend Snippet: (A) HK-2 cells were infected with HCMV strain TR at MOI of 1 and incubated with inhibitors, GM6001, aprotinin, anti-thrombospondin 1(α-TSP), or anti-α v β 6 integrin (α- αvβ6), prior to stimulation with raTGF-β1 at 15 ng/ml (0.6 nM), washed, and TGF-β1 luciferase bioassay performed for active TGF-β1. Results were compared to those from uninfected, unstimulated HK-2 cells (HCMV TR-/raTGF-β1-) as well as HK-2 cells infected with HCMV and stimulated with raTGF-β1 (HCMV TR+/raTGF-β1+). Both GM6001 and aprotinin significantly inhibited active TGF-β1 production. Legend: (**) p<0.05; (***) p<0.01. (B) HK-2 cells were untreated, or were infected with HCMV at MOI of 1 and/or treated with raTGF-β1. Cell lysates were subjected to gelatin zymography (zymogram) and western blotting using anti-MMP-2 (anti-MMP-2). Pro- and active MMP-2 could be detected only in HCMV infected, raTGF-β1 stimulated cells. (C, D) HK-2 cells were treated as in (A), but lysates were either subjected directly to western blotting for TIMP-2, MT3-MMP, MT1-MMP, or actin (C) or incubated with mouse anti-MMP-2 followed by protein A-agarose, and immunoprecipitated proteins subjected to western blotting using rabbit anti-MMP-2, anti-TIMP-2, anti-MT3-MMP, and anti-MT1-MMP. TIMP-2 and MT3-MMP immunoprecipitated with MMP-2 only in HCMV infected, raTGF-β1 stimulated cells. (E) HK-2 cells were transfected with MMP-2 shRNA plasmid (MMP-2), or a control scrambled plasmid (Ctrl). Cells were infected with HCMV strain TR at MOI of 1 and/or stimulated with raTGF-β1 at 15 ng/ml. Supernatants were subjected to luciferase assay for active TGF-β1 (top panel). A portion of the cell pellets were subjected to western blotting for MMP-2, GFP, and actin (middle panel). RNA was extracted from the remainder of the cell pellets and RT-PCR performed for MMP-2 (bottom panel), with results depicted as fold change between raTGF-β1 exposed and non-exposed transfections. These assays showed that MMP-2 shRNA transfection reduced active TGF-β1, MMP-2 protein and mRNA; the control transfections stimulated with raTGF-β1 did induce active TGF-β1, MMP-2 protein and mRNA.

Article Snippet: The following reagents were purchased from commercial vendors: recombinant human active TGF-β1 (raTGF-β1), Quantikine human TGF-β1 ELISA, TGF-β1 blocking antibody (clone 9016) (R&D Systems, Minneapolis MN); luciferase assay reagent, β-galactosidase assay kit (Promega Corp., Madison WI); RNeasy kit (Qiagen, Valencia CA); RT 2 First Strand Kit, SuperArray Human Extracellular Matrix PCR Array, human MMP-2 shRNA kit (SABiosciences, Frederick MD), Cells-to-CT kit (Applied Biosystems, Foster City CA); GM6001, rabbit anti-MMP-2 antibody (AB19167); rabbit anti- MT3-MMP antibody (AB853), mouse anti-α v β 6 blocking antibody (MAB2077Z) (Millipore, Billerica MA); pEF1/myc-his/lacZ plasmid, anti-GFP antibody, AlexaFluor conjugated phalloidin and secondary antibodies, and SuperScript III kit (Invitrogen); Cytogam (CSL Behring, King of Prussia PA); Nucleofector device and transfection kit V (Amaxa, Gaithersburg MD).

Techniques: Infection, Incubation, Luciferase, Zymography, Western Blot, Immunoprecipitation, Transfection, shRNA, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) qPCR analysis of SMG gene expression at distinct developmental stages shows that MT1, MT2, and MT3 increase at E13 when branching morphogenesis begins.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Expressing

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) SMGs were cultured for 36 h with non-silencing (NS) and MT1-, MT2-, and MT3-siRNAs. MT2-siRNA has the most significant effect on epithelial morphogenesis.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Cell Culture

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) Proliferation decreases after MT2-siRNA treatment. The whole mount Ki67 immunostaining in the left hand panels are a single projection. MT2-siRNA increases intracellular collagen IV staining in the epithelium (E, arrows) and mesenchyme (M, arrowheads) in the middle panels. Images are single 2 µM confocal sections.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Immunostaining, Staining

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) SMGs were cultured with MT1- or MT2-siRNAs and NC1 domains for 36 hr. The number of end buds is in the upper graph, and qPCR analysis of MT2 expression is in the lower graph. The reduction in MT2 expression is restored by NC1 domains.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Cell Culture, Expressing

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) SMGs cultured with MT1- or MT2-siRNAs, and HB-EGF (10 ng/ml) or recMT2 (0.05 µg/mL) for 36 h.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Cell Culture

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: Working model showing how MT2-dependent release of bioactive collagen IV NC1 domains regulates protease expression and proliferation during SMG branching morphogenesis. Epithelial MT2-mediated proteolysis of collagen IV releases NC1 domains that signal via β1 integrins and downstream AKT activation to increase MT2 and Col4a2, as well as epithelial proliferation via both FGFR and HBEGF-mediated mechanisms. HBEGF further increases MT2 expression and upregulates endogenous Hbegf, providing further stimulus for rapid SMG branching morphogenesis.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Expressing, Activation Assay

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) qPCR analysis of SMG gene expression at distinct developmental stages shows that MT1, MT2, and MT3 increase at E13 when branching morphogenesis begins.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Gene Expression

Journal:

Article Title: MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

doi: 10.1016/j.devcel.2009.07.016

Figure Lengend Snippet: (A) SMGs were cultured for 36 h with non-silencing (NS) and MT1-, MT2-, and MT3-siRNAs. MT2-siRNA has the most significant effect on epithelial morphogenesis.

Article Snippet: Primary antibodies included rabbit anti-mouse MT1, MT2, and MT3 (Triple Point Biologics, OR), rat anti-mouse perlecan, goat or rabbit anti-mouse collagen IV (both Chemicon, CA), and rat anti-mouse syndecan 1 (BD Pharmingen, CA).

Techniques: Cell Culture

Journal:

Article Title: Cell surface chondroitin sulfate glycosaminoglycan in melanoma: role in the activation of pro-MMP-2 (pro-gelatinase A)

doi: 10.1042/BJ20061176

Figure Lengend Snippet: Melanoma cells (WM1341D) were cultured overnight at 37 °C. In order to inhibit CS modification of the core protein of MCSP, cells were incubated overnight in the presence of 1 mM βDX to block the coupling of CS with the core protein of MCSP. Cell lysates were prepared and cleared by centrifugation at 16000 g for 20 min. The supernatants were collected, precleared and immunoprecipitated (IP) with anti-MCSP or anti-αv integrin antibody (A) or anti-MT3-MMP antibody (B). The immunoprecipitated proteins were released from the beads heated at 90 °C for 5 min under reducing conditions for detecting MCSP and MT3-MMP. αv Integrin and MMP-2 were liberated from beads by incubating at room temperature (25 °C) for 15 min under non-reducing conditions. Proteins were separated on SDS/PAGE. MSCP, αv integrin and MT3-MMP were detected with 9.2.27, AB1930 and AB19088 respectively. MMP-2 was detected by gelatin zymography. Molecular mass markers are shown (kDa). Pro- (P) and active (A) forms of MMP-2 were localized by using conditioned media prepared from ConA-stimulated HT1080 cells. Note the presence of non-specific binding of pro-MMP-2 in the agarose beads used for the immunoprecipitation studies (A). cIgG, control IgG.

Article Snippet: Recombinant TIMP-2 (tissue inhibitor of metalloproteinases-2), rabbit anti-human MT3-MMP (AB19088) and anti-human MMP-2 antibody (AB807), and anti-αv integrin antibodies (MAB1980 and AB1930) were purchased from Chemicon (Temecula, CA, U.S.A.).

Techniques: Cell Culture, Modification, Incubation, Blocking Assay, Centrifugation, Immunoprecipitation, SDS Page, Zymography, Binding Assay