Detection of VEGF protein.

(A) ELISA measurement of secreted VEGF. Levels of VEGF in the CM collected from unstimulated CLL cells were measured using a commercial ELISA kit. The amount of VEGF protein in the samples was calculated using a reference curve established from serial dilutions of rhVEGF protein. Individual histograms represent the means of duplicates. Error bars are standard deviations calculated from 6 measurements in 3 separate experiments using the cells of 3 different patients. (B) Western blot analysis of secreted VEGF. CM was collected from cultures of the PMA-stimulated cells and subsequently concentrated by 10-fold. Each sample (100 μL) was loaded onto a 10% polyacrylamide gel and electrophoresed under nonreducing conditions. Separated proteins were transferred to a nitrocellulose membrane, and VEGF protein was detected with anti-hVEGF mAb and visualized by enhanced chemiluminescence.