ABIN-2 inhibits endothelial cell death. HUVECs were cotransfected with GFP and control vector (CV) or with GFP and vector encoding human ABIN-2. Twenty-four hours after transfection, cells were subject to growth-factor deprivation for 18 hours. (A) Representative fluorescence photomicrographs showing nuclear morphology of GFP-expressing cells cotransfected with CV (upper panel) or ABIN-2 (lower panel). An example of an apoptotic (arrow) and nonapoptotic (arrowhead) transfected cell is indicated. Original magnification, × 200. (B) Apoptotic index of cells was determined as described in “Study design” for HUVECs transfected with CV or ABIN-2 as indicated. Data are presented as means and SEM for 3 independent experiments, ^*P < .01 versus CV. (C) Activated p17/19 caspase-3 was detected by Western blotting in HUVECs transfected with CV or ABIN-2 as indicated. Similar protein loading was confirmed by probing for β-actin and the presence of expressed ABIN-2 confirmed by probing for the FLAG-epitope tag present on the N-terminus of expressed ABIN-2. (D) Survival of cells was determined as described in “Study design” for HUVECs transfected with CV or ABIN-2 as indicated. Data are presented as means and SEM for 5 independent experiments, ^*P < .01 versus CV. (E) Phospho-Ser473 Akt and total Akt were detected by Western blotting in HUVECs transfected with CV or ABIN-2 as indicated. The presence of expressed ABIN-2 confirmed by probing for the FLAG-epitope tag. (F) Survival of HUVEC-transfected CV or ABIN-2 and incubated with control vehicle, 30 nM wortmannin, or 10 μm LY294002 as indicated during growth factor deprivation. Data are presented as means and SEM for at least 3 independent experiments. ^*P < .01 versus CV, control.