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significant increase in phosphorylation

Wound healing assay

HUVECs were grown to confluence in 35-mm dishes coated with gelatin. A linear wound was then made by scratching the monolayer with a plastic tip. The wounded monolayers were washed 3 times with regular medium and incubated in fresh regular medium. At the indicated times, cells were fixed and processed for immunofluorescence staining.

Immunofluorescence analysis

HUVECs were plated in 35-mm dishes at 6 × 104 cells/cm2 (high density) or at 6 × 103 cells/cm2 (low density) and grown for 48 hours. Confluent, sparse, and 3-D collagen cultures were treated accordingly. Cells were washed 3 times with PBS and fixed in acetone/methanol (1:1) at –20°C for 5 minutes. The collagen gels were then dried with Whatman grade 1 paper disks (Maidstone, United Kingdom). The cells were subsequently incubated with polyclonal anti–c-met antibody C-28 (1:100) for 1 hour at room temperature, followed by anti–VE-cadherin antibody (1:100) for 1 hour at room temperature, and finally with a mixture of FITC-conjugated antirabbit IgG antibody (1:100) and TRITC-conjugated antimouse IgG antibody (1:200) for 40 minutes at room temperature. The negative controls consisted of cultures that were incubated only with the secondary antibodies. In the other control experiments, the anti–c-met antibody C-28 was preincubated with the blocking peptide (1:10; wt/wt) for 1 hour at room temperature before being added to the cells. Cells were observed by use of a Nikon (Yokohama, Japan) Eclipse E600 fluorescence microscope and images were recorded with the Perfect Image 5.1 software