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Western blot analysis

Effect of cell density on c-met and VE-cadherin protein levels in monolayer HUVEC cultures

We used double-staining experiments followed by immunofluorescence microscopy to demonstrate that in proliferating sparse HUVECs the c-met and VE-cadherin immunostaining patterns are different from those observed in growth-arrested confluent HUVECs (Figure 1A). Thus, strong immunolabeling with the VE-cadherin–specific antibody was seen in confluent HUVECs at cell-cell contact sites. The immunostaining corresponding to c-met was hardly detectable; only the perinuclear area was very faintly labeled. In contrast, no immunostaining corresponding to VE-cadherin was seen on sparse HUVEC cultures, obtained from the same cells seeded at a lower density (Figure 1A). Conversely, strong immunostaining was seen in sparse HUVECs with the anti–c-met antibody. c-met was distributed rather diffusely over the whole cell surface and did not cluster at precise membrane compartments. No staining was observed when cultures were treated with the anti–c-met antibody that had been preincubated with the blocking peptide, or when the cells were treated with the secondary antibody alone (data not shown).

Figure 1.