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. 2006 Feb 24;394(Pt 3):581–592. doi: 10.1042/BJ20051471

Figure 9. Inhibition of contraction and phosphorylation of MLC and MYPT1 by siRNA-mediated PI3K-C2α silencing, a PI3K inhibitor and a Rho kinase inhibitor.

Figure 9

(A) Inhibition of NA-induced contraction by PI3K-C2α silencing. The VSMCs that had been transfected with either C2α-siRNA or sc-siRNA were stimulated with NA (10 μM), and changes in the planar cell surface area were monitored continuously for 15 min. (B) Quantified results of NA-induced contraction of VSMCs transfected with siRNAs and/or treated with LY at the concentrations indicated. The VSMCs were pretreated with LY or left unpretreated for 30 min, and stimulated with NA (10 μM) for up to 15 min. *, P<0.01 compared with sc-siRNA-treated NA-stimulated control without LY. (C) Ineffectiveness of p110α silencing in inhibiting NA-induced contraction. (D) Inhibition of NA-induced contraction by the Rho kinase inhibitor Y-27632. The VSMCs were pretreated with Y27632 (10 μM) or left unpretreated for 30 min, and stimulated with NA (10 μM) for up to 15 min. *, P<0.01 compared with NA-stimulated Y27632-unpretreated control. (E) Inhibition of NA-induced total MLC phosphorylation. The VSMCs that had been transfected with either C2α-siRNA or sc-siRNA were stimulated with NA (10 μM) for 10 min. *, P<0.01 compared with sc-siRNA-treated non-stimulated control. #, P<0.05 compared with sc-siRNA-treated NA-stimulated control. (F) Inhibition of NA-induced Thr850 MYPT1 phosphorylation by siRNA-mediated PI3K-C2α silencing and/or LY. (G) Inhibition on NA-induced Thr695 MYPT1 phosphorylation of siRNA-mediated PI3K-C2α silencing and/or LY. In (F) and (G), the VSMCs that had been transfected with either C2α-siRNA or sc-siRNA were with LY at the concentrations indicated or left unpretreated for 30 min, stimulated with NA (10 μM) for 10 min, and analysed for Thr850 and Thr695 phosphorylation of MYPT1 by Western blotting using phosphorylation site-specific anti-phospho-MYPT1 antibodies. *, P<0.05 compared with sc-siRNA-treated non-stimulation without LY. #, P<0.05 compared with sc-siRNA-treated, NA-stimulated control. Results are means±S.E.M. of values from 15–45 cells.