GRP-78 inhibits p53-mediated induction of BOK and NOXA and induces phosphorylation of ERK. (A) HUVECs were incubated with increasing concentrations of bortezomib (0.01-1000 ng/mL) for 24 hours. Then the percentage of apoptotic cells was determined by flow cytometry after annexin V and 7-AAD staining. Confluent cells were directly compared with proliferating cells (log phase) and proliferating cells treated with 10nM control protein or GRP-78. *P < .05. (B) Molecular proapoptotic targets of bortezomib and GRP-78 on endothelial cells: p53 phosphorylation and NOXA and BOK expression were analyzed in confluent and log phase growing cells 3 to 12 hours after bortezomib stimulation (10 ng/mL). In contrast to control protein, GRP-78 (10nM) prevented the phosphorylation of p53 (serine 46) and induction of BOK and NOXA. Tubulin-α served as internal loading control. (C) DNA synthesis was studied by the BrdU incorporation assay. In contrast to control cells and cells treated with control protein, 10nM GRP-78 significantly blocked the antiproliferative effect of bortezomib on endothelial cells. Bars represent mean ± SEM. *P < .05. (D) Phosphorylation of the ERK and protein kinase B (AKT) was analyzed on endothelial cells 5 to 90 minutes after treatment with 10 ng/mL bortezomib and GRP-78 or control protein (each 10nM). Tubulin-α served as internal loading control. The presence of GRP-78 resulted in a strong phosphorylation of ERK and to a lesser extent of AKT despite treatment with bortezomib.