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Fig. 1. (A) Specific siRNAs inhibit the expression of p190 isoforms in HUVECs. HUVECs were transfected with the indicated siRNAs targeting either p190A (A1), p190B (B1) or both (A1+B1). After 65 hours, protein extracts were analysed by immunoblotting using specific antibodies. The expression of endogenous β-actin remained unchanged. (Graph) The effect was quantified by measuring the relative amounts of p190 isoforms in each condition. Each bar represents the mean±s.e. of five independent experiments for A1 and B1, and of four independent experiments for A1+B1. (B,C) Specific siRNAs inhibit the expression of p190 isoforms in HUVECs and do not alter the actin cytoskeleton. HUVECs were transfected with the indicated siRNAs, fixed and stained for F-actin and p190A (B) or p190B (C). Scale bars: 10 µm. (D) Silencing p190A and p190B does not modify RhoA activity in HUVECs. Cells treated with 2 µg/ml of lysophosphatidic acid (LPA) for 15 minutes or transfected with the indicated siRNAs were lysed and active RhoA GTPase was affinity-precipitated with GST-RBD-rhotekin, eluted from the beads and analysed by immunoblotting using anti-RhoA antibodies. For each condition, a fraction of the lysate was run to monitor the RhoGAP knockdowns and the amount of GTPase before precipitation. The graph shows the quantification of bands from two independent experiments; error bars represent the mean±s.e. RhoA activity was quantified in comparison to the negative control (control-siRNA-transfected cells). In p190-KD cells, basal levels of RhoA were not altered. LPA, which is a potent activator of RhoA, was used as a positive control in this experiment.
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