Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: Endothelial cell tubule formation assay. BME cells (3 × 105/ml) were seeded onto type I collagen gels in medium supplemented with 10% calf serum. On the following day, the medium was aspirated and replaced with conditioned serum-free medium derived from cocultures of G8 myoblast monolayers and TA3wt (A) TA3sCD44 (B), TA3sCD44/MMP-9-CD44fp (C), or TA3sCD44/MMP-9v5 (D) cells. A total of 30 μg/ml of pan specific anti-TGF-β (E) or anti-bFGF (F) antibody were added to the TA3wt/G8-conditioned coculture medium prior to use in the assay. Tubules are indicated by arrows. Bar, 140 μm.

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Tube Formation Assay, Derivative Assay

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: CD44-anchored MMP-9 activates TGF-β. (A) The ability of TA3 transfectant-G8 myoblast coculture media to stimulate TMLC luciferase activity is shown. Conditioned coculture media tested are indicated. (B) Pan-specific TGF-β antibody (30 μg/ml) or specific antibodies against TGF-β1 (100 ng/ml), TGF-β2(100ng/ml), or TGF-β3(100 ng/ml) were used to determine the activity of TGF-β isoforms in the coculture media. One unit of luciferase activity corresponds to the activity produced by 5 pg of purified human TGF-β1 (R & D).

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Activity Assay, Produced, Purification

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: TGF-β is activated by purified MMP-9. (A) TMLC luciferase activity induced by concentrated COS cell supernatants containing the indicated latent TGF-β isoforms following incubation with the indicated purified AMPA-activated MMPs. Comparable amounts of latent TGF-β1, TGF-β2, and TGF-β3 were present in the supernatants as assessed by both Western blot analysis and TMLC-luciferase induction following heat treatment (80°C for 5min) (B) TMLC luciferase activity induced by affinity-purified TGF-β2 following incubation with the indicated purified activated MMPs. Activity is expressed in relative light units (RLU), in which 800 RLU corresponds to the luciferase activity generated by 1 pg of purified human TGF-β1 (R & D). (C) TGF-β is proteolytically cleaved by MMP-9 and MMP-2. Purified v5-tagged TGF-β2 (lane 1) was incubated with protein-A Sepharose-bound, AMPA-activated MMP-9 (lane 2), MMP-2 (lane 3), and MMP-3 (lane 4) for 90 min at 37°C. Following incubation, the supernatants were separated from the beads, subjected to SDS/12% PAGE, transferred to Hybond-C membranes, and blotted with anti-v5 antibody. (D) MMP-2/CD44 fusion protein expression promotes TGF-β activation in TA3sCD44 cells. TMLC luciferase assays were performed with serum-free coculture media from TA3sCD44 cells transiently transfected with the indicated cDNAs.

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Purification, Luciferase, Activity Assay, Incubation, Western Blot, Affinity Purification, Generated, Expressing, Activation Assay, Transfection