mouse anti adam 9 monoclonal antibody  (R&D Systems)

Journal:

Article Title: Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs

doi: 10.1093/emboj/cdf532

Figure Lengend Snippet: Fig. 4. Expression of ADAMs in human keratinocytes. (A) Immuno fluorescence staining of collagen XVII and ADAMs in the skin. Collagen XVII antibody NC16A produced a linear signal at the basement membrane zone (a). TACE (b) and ADAM-9 (c) immunoreactivity was stronger in the lower epidermis, in the neighborhood of collagen XVII, whereas ADAM-10 immunoreactivity (d) was distributed throughout the entire epidermis. Scale bar: 250 µm. (B) Immunoblot of keratinocyte extracts with antibodies to the ectodomains of TACE and ADAM-9, and the endodomain of ADAM-10. TACE and ADAM-9 antibodies recognized two distinct protein bands of ∼120 and 85 kDa, and 110 and 79 kDa, respectively, representing the proform and the active form of the enzymes. ADAM-10 antibody recognized one band of ∼60 kDa, corresponding to the active form. (C) Activation of TACE in keratinocytes was inhibited by a furin inhibitor, but not by hydroxamates. Keratinocytes were cultured for 24 h in serum-free medium with or without 50 µM furin inhibitor decanoyl-RVKR-chloromethyl ketone or 25 µM hydroxamate BB 3103. Keratinocyte lysates were immunoblotted with TACE antibodies. The furin inhibitor, but not BB 3103, prevented the conversion of pro-TACE to active TACE. Molecular weight markers are shown on the left.

Article Snippet: As first antibodies, collagen XVII antibody NC16A, TACE antibodies against the extracellular pro- and metalloproteinase domains (Santa Cruz Biotechnology) and the intracellular C-terminus (Chemicon International, Hofheim, Germany), and antibodies to ADAM-10 endodomain (Chemicon) and ADAM-9 ectodomain (R&D Systems, Wiesbaden, Germany) were used.

Techniques: Expressing, Fluorescence, Staining, Produced, Membrane, Western Blot, Activation Assay, Cell Culture, Molecular Weight

Journal:

Article Title: Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs

doi: 10.1093/emboj/cdf532

Figure Lengend Snippet: Fig. 5. Transient transfections of HaCaT cells with cDNAs for TACE, ADAM-9 and ADAM-10. (A) Immunofluorescence staining of transfected cells. Cells transfected with empty vector (control) remained negative, but ADAM-transfected cells showed strong positive signals. Scale bar: 100 µm. (B) HaCaT cells were transfected with different concentrations of cDNA for TACE (upper panel), ADAM-10 (middle panel) and ADAM-9 (lower panel) and shedding of collagen XVII was assessed with immunoblotting with a mixture of antibodies Endo-2, NC16A and Ecto-1. Densitometric analysis of the signals (expressed as a percentage of the control ± SD; n = 3) showed a dose-dependent increase of ectodomain shedding, with a concomitant decrease of full-length collagen XVII.

Article Snippet: As first antibodies, collagen XVII antibody NC16A, TACE antibodies against the extracellular pro- and metalloproteinase domains (Santa Cruz Biotechnology) and the intracellular C-terminus (Chemicon International, Hofheim, Germany), and antibodies to ADAM-10 endodomain (Chemicon) and ADAM-9 ectodomain (R&D Systems, Wiesbaden, Germany) were used.

Techniques: Transfection, Immunofluorescence, Staining, Plasmid Preparation, Western Blot

Journal:

Article Title: Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs

doi: 10.1093/emboj/cdf532

Figure Lengend Snippet: Fig. 8. Increased collagen XVII shedding correlates with decreased cell motility. In wound closure assays cells migrate from the edges of a ‘scrape wound’ in the monolayer (interrupted lines) to cover the denuded space (between the lines). Scrape wounds were made in serum-free cultures, and after washing the cells were allowed to re-epithelialize for 22 h at 37°C. Micrographs of non-fixed cell layers at 0, 11, 16 or 22 h after wounding are shown. Scale bars: 200 µm. (A) Transfection of HaCaT cells with ADAM cDNA negatively regulated cell motility. Transfections were with 15 µg of empty vector (control), 15 µg of TACE, 15 µg of ADAM-10, or 15 µg of ADAM-9 cDNA. (B) Addition of purified collagen XVII ectodomain to normal human keratinocytes inhibited cell motility. After wounding, 1 nM collagen XVII ectodomain was added to the culture, and the cells were permitted to migrate into the denuded space for 16 h. Ectodomain-treated cells did not cover the denuded area as efficiently as control cells.

Article Snippet: As first antibodies, collagen XVII antibody NC16A, TACE antibodies against the extracellular pro- and metalloproteinase domains (Santa Cruz Biotechnology) and the intracellular C-terminus (Chemicon International, Hofheim, Germany), and antibodies to ADAM-10 endodomain (Chemicon) and ADAM-9 ectodomain (R&D Systems, Wiesbaden, Germany) were used.

Techniques: Transfection, Plasmid Preparation, Purification