Shc is required for VEGF-induced EC survival but not migration toward VEGF or proliferation. (A) HUVECs were serum starved for 24 hours with or without 100 ng/mL of VEGF to induce apoptosis. Lysates were immunoblotted for cleaved caspase 3 and GAPDH as a loading control. Survival was quantified by comparing the amount of cleaved caspase 3 present in lysate. Values shown are means ± SEM (n = 4 independent experiments). (B) Chemotaxis toward the VEGF gradient was measured using Boyden chambers containing 100 ng/mL of VEGF or vehicle in the lower well. After 4 hours of migration, cells that had migrated to the underside of the membrane were counted using an inverted microscope. Five random fields were imaged per filter. Values shown are means ± SEM (n = 3 independent experiments, 2 filters per condition per experiment). *P < .05. (C) EC proliferation was assayed in the P5 mouse retina. EdU reagent was injected intraperitoneally and 2 hours later retinas were harvested. Retinas were stained with isolectin (green) to mark ECs, DAPI (blue) to mark all cell nuclei, and EdU (red) to mark proliferating nuclei. Proliferation of ECs was quantified by counting the number of isolectin/EdU⁺ nuclei and dividing by the number isolectin/DAPI⁺ nuclei. Values shown are means ± SEM (Shcflox/flox, n = 5 and Shcflox/flox;Tie2-Cre, n = 8).