FIGURE 8.

R-Ras does not alter receptor expression or transferrin internalization. A–E, R-Ras has no effect on cell surface and total expression of VEGFR2, neuropilin 1, and ephrin B2. A, cells without VEGF stimulation were incubated with anti-VEGFR2 antibody at 4 °C for immunofluorescence staining of cell surface, and fixed with paraformaldehyde. Scale bar = 25 μm. B, cell surface proteins were biotinylated, and cell lysate was prepared without VEGF stimulation or biotin stripping. The cell surface proteins were isolated from the lysate by avidin column, and anti-VEGFR2 Western blotting was performed to quantify the surface VEGFR2 levels. An aliquot of cell lysate was loaded directly onto the gel to determine total VEGFR2 and GAPDH (Input). C, quantification of surface and total VEGFR2 normalized to GAPDH, presented as the levels relative to mock control cells. D, the cell surface expression levels of neuropilin 1 and ephrin B2 were determined for R-Ras38V or mock control cells. The total expression levels of these proteins were also determined using the pre-avidin column lysate. E, similar analyses were conducted for R-Ras-silenced ECs. No significant differences were found in R-Ras38V or R-Ras shRNA-transduced cells compared with control cells. NC, negative control shRNA. F, Alexa Fluor 555-conjugated transferrin was allowed to internalize into the cells at 37 °C for 5 min. The level of transferrin internalization was quantified by average integrated fluorescence signal intensity per cell and is presented as relative values.