VEGF increases both protein synthesis and mRNA for Mcl-1 and XIAP in CLL B cells. (A) The blots illustrate the increase in both Mcl-1 and XIAP in CLL B cells exposed to VEGF. CLL B cells were cultured in serum-free RPMI for 24 hours and then stimulated with VEGF₁₆₅ (50 ng/mL) for 30 minutes prior to being lysed. Protein extracts (50 μg) were subjected to SDS-PAGE and then transferred to nitrocellulose. The blot was then probed with the antibody of interest. Data shown are representative of 1 of 5 different experiments. The results were consistent and reproducible. (B) VEGF stimulates increased expression of Mcl-1 and XIAP mRNA. Freshly isolated CLL B cells were pretreated with actinomycin D (1000 nM) for 24 hours prior to VEGF₁₆₅ (50 ng/mL) stimulation for an additional 30 minutes. RNA was then isolated and RT-PCR was used to determine Mcl-1, XIAP, and Bcl-2 mRNA levels. The far right lane in panel B indicates our blank control for this set of experiments. The ratio (mRNA/actin) is used to determine the incremental mRNA of interest based on densitometry. The value from the table indicates the fold mRNA of interest based on densitometry. Each value is normalized to the control. This experiment has been repeated twice on 2 different patients with CLL. Act D indicates actinomycin D.