Fig. (8) Western blot validation of iTRAQ results. Randomly selected proteins significantly regulated in tianma-stimulated rat aortas compared with controls. Rats were treated, proteins extracted and western blot performed as described in materials and methods. A: Serpinh1, Anxa2 and Gstm2 expressions were up-regulated, while Vcl and Des expressions were down-regulated. Gapdh, that did not show any change in the iTRAQ data set, was used as reference protein. B: Quantitative analyses of the western blots shown in A. Western blots were performed at least four times for statistical quantification and analyses (n=4). Values (= relative protein expression) represent the ratio of densitometric scores for the respective western blot products and statistical error was indicated as mean ± SD (*P < 0.05, compared with controls) using the Gapdh bands as reference. C: The histogram indicates a similar close relationship between iTRAQ and western blot expression ratios. Tianma-stimulated and control aorta iTRAQ expression ratios from selected proteins were consistent with the western blot results and thus validated a strong agreement in the expression data.