Fig. 6.

Verification analyses of differential expression of identified proteins. A, validation of the iTRAQ results of selected proteins by immunoblotting using protein extracts from transfected bladder cancer cells. The results verified differential regulation of these proteins upon KiSS-1 transient overexpression. Parallel to KiSS-1 overexpression, decreased expression was found for Filamin A, and increased expression was observed for Ezrin. Antibodies were accepted as displaying a single predominant band at the expected molecular weights; α-tubulin was used as the loading control. B and C, representative immunohistochemistry expression patterns of Filamin A in non-invasive and invasive bladder tumors contained in tissue arrays. High expression levels of Filamin A were observed in non-invasive superficial lesions as compared with invasive bladder tumors. There was a significant difference regarding the expression of Filamin A regarding tumor stage (p < 0.0005) (original magnifications in C and D, ×200). D, Kaplan-Mayer curve survival analysis indicating that a decreased protein expression of Filamin A measured by immunohistochemistry on tissue arrays was significantly associated with poor disease-specific survival (p = 0.001).