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. 2020 Oct 16;21(20):7679. doi: 10.3390/ijms21207679

Figure 4.

Figure 4

NOX1-mediated ROS generation is required for Y-27632-induced PAK1 activation. (A) PC12 cells were treated with 25 μM Y-27632 for the indicated times in the presence or absence of ROS scavengers, such as 5 mM NAC and 500 μM trolox, and cell lysates were then immunoblotted with antibodies against phosphorylated PAK1 (at Ser199/204), PAK1, and GAPDH. The p-PAK1 protein expression level was normalized to that of total PAK1 and is shown in the right panel. (B) PC12 cells were treated with 25 μM Y-27632 for the indicated times in the presence or absence of 10 μM DPI, and the cell lysates were then immunoblotted with antibodies against phosphorylated PAK1 (at Ser199/204), PAK1, and GAPDH. The p-PAK1 protein expression level was normalized against PAK1 and is shown in the right panel. (C) PC12 cells transfected with control shRNA or NOX1 shRNA were treated with 25 μM Y-27632, and the cell lysates were then immunoblotted with antibodies against phosphorylated PAK1 (at Ser199/204), PAK1, and GAPDH. The p-PAK1 protein expression level was normalized to that of total PAK1 and is shown in the right panel. Note that scavenging ROS with NAC or trolox, inhibiting NOX1 with DPI, and knocking down NOX1 all inhibited Y-27632-induced PAK1 phosphorylation. The data represent the means ± S.E.M. of at least three independent experiments. * p < 0.05; ** p < 0.01.