Fig. 9.

Internalization and activation status of VEGFR2 in response to VEGF₁₆₅a. (a) Time course of VEGF₁₆₅a-TMR binding to NLuc-VEGFR2. Furimazine was added 5 mins prior to VEGF₁₆₅a-TMR. (b) Time course of the internalization of VEGF₁₆₅a-TMR (red circles) and activated VEGFR2 (stained with antibodies targeting phosphate moieties indicative of VEGFR2 activation) within the same cells. 3 nM VEGF₁₆₅a-TMR was added at set time points (1–120 min). Cells were fixed using 3% PFA/PBS, permeabilised (0.025% Triton-X-100/PBS) and labeled with a rabbit antibody targeting pY1175 (purple triangles) or pY1214 of VEGFR2 (black circles). Cells were imaged using a IX Micro widefield platereader. Data are expressed as a percentage of the peak responses at 20 min and represent mean ± S.E.M from 3 independent experiments. (c) Timecourse of 3 nM VEGF₁₆₅a induced internalization of VEGFR2-HaloTag. Cells were labeled with 0.5 μM HaloTag-TMR fluorescent ligand as described under Methods. Images were analysed and data expressed as described for (b). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)