Fig. 1.

Effects of vandetanib on receptor phosphorylation. A, T98G cells were seeded at 60% confluence and allowed to attach. The cells were then serum-starved for 24 h and pretreated with 2 μM vandetanib for 2 h, and then left untreated or treated with 50 ng/ml EGF for 30 min. Cell extracts were prepared, and equal amounts of protein (30 μg/lane) were separated by SDS-PAGE analysis and subjected to Western blot analysis with the indicated primary antibodies. B, T98G and A172 cells were seeded at subconfluence and incubated overnight at 37°C. Then, the cells were serum-starved for 24 h and pretreated with 0 to 2 μM vandetanib for 2 h and subsequently left untreated or treated with 50 ng/ml EGF. Cell extracts were prepared, and equal amounts of protein were separated and subjected to Western blot analysis with phospho-EGFR antibody (Tyr845). The blots were subsequently stripped and reprobed against total EGFR. C, T98G cells were seeded as mentioned above. After 24 h of serum starvation, cells were pretreated with 2 μM vandetanib for 2 h and then left untreated or treated with 50 ng/ml EGF or 50 ng/ml VEGF for 30 min. Western blot analysis was performed as described in Materials and Methods and probed with indicated antibodies. D, after overnight attachment, T98G cells were serum-starved for 24 h and pretreated with 0 to 2 μM vandetanib for 2 h and then left untreated or treated with 50 ng/ml VEGF. Cell extracts were prepared, and equal amounts of protein were separated and subjected to Western blot analysis with phospho-VEGFR-2 antibody. The blots were subsequently stripped and reprobed against total VEGFR-2. E, T98G cells were serum-starved for 24 h, pretreated with 2 μM vandetanib for 2 h, and then left untreated or treated with 50 ng/ml EGF, 50 ng/ml VEGF, and 50 ng/ml PDGF for 30 min. Cell extracts were prepared, and equal amounts of protein (30 μg/lane) were separated by SDS-PAGE analysis and subjected to Western blot analysis with the phospho-PDGFRβ antibody. Subsequently, the blot was stripped and reprobed with total PDGFRβ antibody. F, T98G cells were seeded at subconfluence and incubated overnight at 37°C. Then, the cells were serum-starved for 24 h and pretreated with 0 to 2 μM vandetanib for 2 h and left untreated or treated with 50 ng/ml PDGF. Cell extracts were prepared, and equal amounts of protein were separated and subjected to Western blot analysis with phospho-PDGFRβ antibody. The blots were subsequently stripped and reprobed against total PDGFRβ.