Figure 8.
Silencing clathrin expression inhibits VEGFR-2 phosphorylation and signaling in VEC-null cells. VEC-null and -positive cells were transfected with Stealth siRNA-targeting mouse clathrin heavy chain. Two oligonucleotides (a and b; respective sequences are reported in Materials and methods) were used that target two independent sequences of the clathrin heavy chain mRNA. Negative Stealth siRNA duplex was used as a control. After 72 h, cells were treated with VEGF or control medium for 10 min. They were then extracted and processed for Western blotting. Clathrin siRNA reduced clathrin heavy chain levels by ∼75 (oligonucleotide a) and 90% (oligonucleotide b) in both cell types. VEGFR-2 phosphorylation at tyrosine 1214 and p44/42 MAPK phosphorylation in response to VEGF were strongly inhibited in VEC-null cells, whereas these parameters were only barely affected in VEC- positive cells. Comparable results were obtained for PY1054/59–VEGFR-2 (not depicted). The graphs show the quantification of these effects as means ± SD (error bars) of five independent experiments. Fold increase after VEGF is shown for PY1214–VEGFR-2 and phospho-p44/42 MAPK. *, P ≤ 0.01 versus negative siRNA by variant analysis and the Dunnet test.