Normal splenic B and CLL B cells express differences in VEGF receptor autophosphorylation status. Protein and phosphorylation level of VEGF-R1 and VEGF-R2 in CLL B cells and splenic B cells are shown. (A) Isolated CLL B cells or spleen cells from a healthy donor were cultured in serum-free RPMI for 24 hours. Lysates (500 μg) were prepared and immunoprecipitated with either anti–VEGF-R1 or anti–VEGF-R2 antibody and then transferred to nitrocellulose. Blots were probed with phosphotyrosine antibody. Note the protein levels of VEGF-R1 and VEGF-R2 were approximately equivalent in both cell types, but the levels of spontaneous phosphorylation for VEGF-R1 and VEGF-R2 in spleen cells are much lower than in CLL B cells. (B) Densitometry was used to normalize the phosphorylated VEGF-R (p-VEGF-R) levels in the immunoblot and shows that the CLL B cells still retain a higher overall phosphorylation value for both VEGF-R1 and VEGF-R2 when compared with spleen cells.