FIGURE 4.

Reverse-mode NCX activity is required for ERK1/2 activation by a peptide agonist of PAR-1. A, HUVECs loaded with BAPTA-AM were challenged with the PAR-1 peptide agonist TFLLR-NH₂ (25 μm for 5 min). ERK1/2 and PLCβ3 activation was determined as in Fig. 1A (n = 3). B, HUVECs, preincubated with SEA0400, were stimulated with TFLLR-NH₂ (25 μm for 5 min). ERK1/2 and PLCβ3 phosphorylation was determined as before (n = 3). C, HUVECs were preincubated with SN-6 and stimulated with TFLLR-NH₂ for the indicated times. D, HUVECs transfected with NCX1 or control siRNA as in Fig. 2F were stimulated with TFLLR-NH₂ (25 μm for 5 min); ERK1/2 and PLCβ3 activation was evaluated as in Fig. 1A. NCX1 protein knockdown for this experiment is shown in Fig. 2H, in cells transfected in parallel. E, HUVECs were treated with ouabain (100 μm for 1 min) prior to stimulation with TFLLR-NH₂ (5 μm for 2 min). Where indicated, samples were preincubated with SN-6 (10 μm) for 30min. For A–F, n = 3.