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. Author manuscript; available in PMC: 2011 May 18.
Published in final edited form as: Dev Cell. 2010 May 6;18(5):713–724. doi: 10.1016/j.devcel.2010.02.016

Figure 3. VEGFR-2 Trafficking in Synectin−/− and MyosinVI−/− Primary Endothelial Cells.

Figure 3

(A) VEGFR-2 internalization is similar in synectin and myosin-VI+/+ and −/− endothelial cells: Confluent, serum starved cells were surface labeled with biotin and stimulated with VEGF-A. After stripping remaining cell surface biotin, cell lysates were precipitated with NeutrAvidin beads and Western blots probed for VEGFR2 to determine internalized VEGFR2. See also Fig S2.

(B–D) VEGFR2 trafficking: Serum starved synectin+/+ and −/− or myosin-VI-+/+ and −/− AEC cells labeled with anti-VEGFR2(green) were treated with VEGF-A for 5–30min. and then fixed, permeablized, labeled with anti-EEA1(red) and visualized using immunofluorescent microscopy. Quantification of VEGFR2/EEA1 co-localization at various time points is shown for synectin+/+ and −/− AEC in panel C and myosin-VI+/+ and −/− AEC in panel D. (Mean±SD, * P < 0.05)

(E) Movement of VEGFR-2 containing vesicles in synectin−/− AEC: Serum starved cells labeled with anti-VEGFR-2 were treated with VEGF-A and the labeled vesicles tracked by time-lapse microscopy. Images were acquired every 30 seconds for 30 minutes and vesicle mean speed and increment were determined using Image J. (Mean±SD, * P < 0.05). Sere also Fig S3