Identification of GRP-78 in the prostate carcinoma cell supernatant. (A) SEC of PC-3 supernatants was performed, and single fractions were tested on HUVECs in the WST-1 cell viability assay. All fractions were tested in the presence of 10 ng/mL bortezomib in direct comparison with unfractionated supernatant (SN). Untreated HUVECs served as positive control (co.+) and HUVECs stimulated with bortezomib in the absence of tumor supernatant as negative control (co.−). The scale indicates the molecular weight (kDa) of the individual fractions. A fraction with the molecular size of approximately 70 kDa showed the strongest inhibitory effect on endothelial cell growth. (B) IEC of PC-3 supernatants was performed, and the fractions were analyzed in HUVECs in the WST-1 assay. The scale indicates the approximate pI of the various fractions. Only a very acidic fraction with a pI of less than 4.5 was able to block the antiangiogenic effect on HUVECs. Bars represent mean ± SEM. *P < .05. (C) GRP-78 was the main compound in both biologically active fractions (Fr.8 from SEC and Fr.15 from IEC). Fractions were analyzed with recombinant GRP-78 (100-nM solution) either by SDS-PAGE stained with Coomassie (left panels) or by Western blot (right panel) to estimate GRP-78 concentrations and purity.