Figure 5

Rac1 activity and oxidative stress are increased in p190B transgenic tumors. (a) G-LISA assays were used to quantify the levels of active RhoA, Rac1, and Cdc42 in rtTA (n =11) and p190B (n =11) tumors. A Mann-Whitney rank sum test was used to compare the means, and error bars represent standard error of the mean. Rac1 activity was elevated, however, RhoA and Cdc42 activities were not statistically significantly different between the two groups. (b) An OxyBlot assay was performed to detect protein carbonylation as an indirect measure of ROS-mediated oxidative stress. Western blot shows an increase in total carbonylated proteins after dinitrophenylhydrazine (DNP) derivitization in p190B expressing tumor lysates compared to control lysates. Each lane represents equal amounts of protein pooled from 11 tumors per genotype. DNP protein standards were included for size comparison. β-actin is shown as a loading control. Densitometry value represents the fold change in carbonylated proteins in p190B tumors relative to control tumors. Control tumor values were set to 1 and samples were normalized to β-actin. (c) Western blotting to detect expression of total and phosphorylated forms of downstream effectors of Rac1. β-actin is shown as a loading control. Each lane represents equal amounts of pooled protein from 11 tumor lysates per genotype. Densitometry values represent the fold change in protein expression in p190B transgenic tumors relative to control tumors. Control tumor values were set to 1 and samples were normalized to β-actin.