Fig. 1

The relationship between H-ras ^v12 and Lu/BCAM. (A) Expression levels of Lu/BCAM and Ras in human bladder uroepithelial cell line - E6RC harboring an inducible H-ras ^V12 oncogene in the presence or absence of IPTG (5 mM) for 48 h were measured by immunoblotting. β-actin was used as an equal loading control. (B) The mRNA expression level of Lu/BCAM in five bladder cancer cell lines, i.e. immortalized (E6), early- (RT4), middle- (TSGH8301) and late-stage (TCCSUP and J82), was evaluated by real-time PCR. PPIA (peptidyl prolylisomerase A) was used as internal control to normalize the expression of Lu/BCAM. (C) The pGL3-Lu reporter plasmid together with pSG5ras (expressing H-ras ^V12), pGL3basic and pBSSK⁺ were co-transfected into HEK293 cells and luciferase activity was determined using Dual-Luciferase™ Reporter assay system. The pRL-TK vector was used as an internal control for normalization. (D) Localization of Lu/BCAM protein in bladder cancer specimen by IHC staining using monoclonal anti-Lu antibody. Membranous expression of Lu/BCAM in the basal compartment of bladder carcinoma is indicated by arrow, while Lu expression in the endothelium and smooth muscle cells is shown by arrowhead (magnification: 300X; Olympus). (E) Localization of Lu/BCAM protein in TSGH8301 cells was detected by M2-flag monoclonal antibody using immunofluorescent assay under fluorescence microscopy (magnification: 400X; Olympus). a: Cell only; b: Cells transfected with vector plasmid pTRE2Hyg; c: Cells were co-transfected with plasmids of pTRE2Flag-Lu and pTet-Lac-Hyg, and then treated with doxycycline (1μg/ml); d: Cells received the same treatment as “c” except without doxycycline. Arrow points Lu protein