Supplementary Fig

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1 Supplementry Fig. 1 * * Mem Cyt Mem Cyt Supplementry Fig.1 Specificity of nti-tie2 ntiodies. HUVECs were homogenized nd centrifuged t g to otin totl memrne (Mem) nd cytosolic (Cyt) frctions. Smples from ech frction were resolved y SDS-PAGE nd Western lots were performed using either () mouse monoclonl nti-tie2 ntiodies, or () got polyclonl nti-tie2 ntiodies. * Indictes the position of mture Tie-2. Numers indicte moleculr mss in kd shown on the left.

2 Supplementry Fig. 2 Supplementry Fig. 2 Vlidtion of the Tie-2 leling strtegy. 293F cells nd 293F cells stly expressing Tie-2 were grown on glss coverslips tht were pre-coted with poly-l-lysine. The cells were fixed s descried for HUVECs in Methods. Both 293F cells nd 293F cells expressing Tie-2 were treted with mouse monoclonl nti-tie2 ntiodies (10μg/ml) followed y got nti-mouse secondry ntiodies conjugted to 0.8nm gold prticles. The sizes of the gold prticles were enhnced with silver. Cells were viewed using the Hitchi S-5200 scnning electron microscope. Bcksctter imges showing the surfce of () 293F cells, nd () 293F cells expressing Tie-2.

3 Supplementry Fig. 3 Supplementry Fig.3 Vlidtion of the Tie-2 leling strtegy. 293F cells nd 293F cells stly expressing Tie-2 were grown on glss coverslips tht were pre-coted with poly-l-lysine. The cells were fixed s descried for HUVECs in Methods. Both 293F cells nd 293F cells expressing Tie-2 were treted with got polyclonl nti-tie2 ntiodies (2μg/ml) followed y rit nti-got secondry ntiodies conjugted to 0.8nm gold prticles. The sizes of the gold prticles were enhnced with silver. Cells were viewed using the Hitchi S-5200 scnning electron microscope. Bcksctter imges showing the surfce of () 293F cells, nd () 293F cells expressing Tie-2.

4 Supplementry Fig. 4 Supplementry Fig.4 Bckground control imges for Tie-2 leling. HUVECs were fixed s descried in Methods nd treted with secondry ntiodies conjugted to 0.8nm gold prticles followed y silver enhncement. Cells were imged using the Hitchi S-5200 scnning electron microscope. Bcksctter imges showing ckground leling using () rit nti-got secondry ntiodies, nd () got nti-mouse secondry ntiodies.

5 Supplementry Tle 1: Quntifiction of Tie-2 Clusters on the HUVEC Plsm Memrne Cluster Size Mouse monoclonl Got polyclonl After exmining 70μm 2 of the HUVEC plsm memrne, the numer of times cluster size ppered ws counted for ech nti-tie2 ntiody. Cluster size is defined s the numer of ggregted silver prticles. Bold numers refer to the numer of silver prticles in the cluster; cluster size of 1 refers to single isolted silver prticle, 2 refers to cluster contining two silver prticles, 10 refers to cluster contining 10 silver prticles.

6 Supplementry Fig. 5 Supplementry Fig.5 Intrcellulr leling of R5. HUVECs were grown s monolyer to confluence, fixed, permeilized, nd treted with mouse monoclonl nti-r5 ntiodies (BD Biosciences, 1:50 dilution) followed y donkey nti-mouse secondry ntiodies conjugted to 0.8nm gold prticles. The sizes of the prticles were enhnced with silver. The cells were emedded nd sectioned (90nm thick). Sections were stined with urnyl cette nd imged using the Hitchi H-7000 trnsmission electron microscope.

7 Supplementry Fig. 6 Supplementry Fig.6 Vlidtion of the Ang-1 leling strtegy. 293F cells expressing Tie-2 were grown on glss coverslips tht were pre-coted with poly-l-lysine. Cells were incuted on ice for 90 min in medi with or without Ang-1 (800ng/ml). The cells were wshed, fixed, nd treted with mouse monoclonl nti-ang1 ntiodies (1μg/ml) followed y donkey nti-mouse secondry ntiodies conjugted to 0.8nm gold prticles. The sizes of the gold prticles were enhnced with silver. Cells were imged using the Hitchi S-5200 scnning electron microscope. Bcksctter imges of () Ang-1 on the cell surfce, nd () ckground control.

8 Supplementry Fig. 7 Supplementry Fig.7 Bckground control for Ang-1 leling in HUVECs. Cells were incuted on ice for 90min in the sence of Ang-1. The cells were wshed, fixed, nd exposed to mouse monoclonl nti-ang1 ntiodies (1µg/ml) followed y donkey nti-mouse secondry ntiodies conjugted to 0.8nm gold prticles. The sizes of the gold prticles were enhnced with silver. Cells were viewed using the Hitchi S-5200 scnning electron microscope.