Figure 4. Inhibition of PKD substrate phosphorylation by CRT5 and PKD knockdown.

(A) HUVECs were transfected with 200 nM siRNA targeting PKD1 and PKD2, or 400 nM of a control siRNA (Scr). At 48 h after transfection, cells were then treated for a further 10 min with no addition (C) or with 25 ng/ml VEGF (V). In parallel cell cultures, confluent HUVECs were pre-incubated for 30 min with either vehicle (Cont; 0.1% DMSO) or 5 μM CRT5, followed by 10 min stimulation with vehicle (C) or 25 ng/ml VEGF (V). Cell lysates were then immunoblotted with antibodies against total and phosphorylated HSP27 (Ser⁸²). Knockdown of PKD1 and PKD2 was confirmed using an antibody that recognizes both PKD1 and PKD2 [10]. Results from at least three independent experiments were quantified by scanning densitometry and are presented as the ratios of phospho-HSP27 to total HSP27 (means±S.E.M., arbitrary units). A representative blot corresponding to the quantified results is shown above the histogram. Two-way ANOVA indicated a significant interaction and treatment effects (P<0.05 and P<0.0001 respectively); *P<0.05 compared with VEGF plus Scr siRNA or VEGF plus vehicle as determined by Bonferroni's post hoc test. (B) HUVECs treated as detailed in (A) were immunoblotted with antibodies against total or phosphorylated CREB. Two-way ANOVA indicated a significant interaction, inhibitor and treatment effect (P<0.005); *P<0.01 compared with VEGF control siRNA or VEGF vehicle respectively as determined by Bonferroni's post hoc test. (C) HUVECs treated as detailed in (A) were immunoblotted with antibodies against total or phosphorylated HDAC5. Two-way ANOVA indicated a significant inhibitor and treatment effects (P<0.05 and P<0.001 respectively);*P<0.05 compared with Scr siRNA plus VEGF or vehicle plus VEGF as determined by Bonferroni's post hoc test.