The membrane was then washed three times in TBST, incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 3000, Bio-Rad Laboratories) for 1 h at room temperature and washed again. Proteins were visualized using

the SuperSignal West Pico Chemiluminescent Substrate (Pierce). To ensure equal amounts of FAK in all samples, the membrane was stripped and reprobed with rabbit anti-FAK antibody C-309 (1 : 200 in blocking buffer, Santa Cruz Biotechnologies). Digital images of the membrane were analyzed using imagej software (NIH, Bethesda, MD, http://rsb.info.nih.gov/ij/). Data were analyzed by one-way anova with the Newman–Keuls multiple comparison post test using the graphpad prism version 5.02 (GraphPad Software,

San Diego, CA). BMS-777607 in vitro Differences with P-values <0.05 were considered statistically Paclitaxel cost significant. The expression of EpoR in nonerythropoietic tissue is debated (Ghezzi et al., 2010; Sinclair et al., 2010; Swift et al., 2010; Xiong et al., 2010). A prediction for our hypothesis was, however, that EpoR, as part of the heteromer with CD131, is expressed in the bladder epithelium. We therefore tested the bladder epithelial cell lines and primary bladder epithelial cells used in our cell infection model for EpoR expression. We could detect low constitutive levels of EpoR-specific mRNA in all three bladder cell types investigated in this study (Fig. 1) as well as in the monocytic cell line THP-1. Discrepancies among the findings these reported by others might result from the different sensitivities of methods or interpretation criteria (Ghezzi et al., 2010). Contact between E. coli and bladder epithelial cells induces a general inflammatory response. In other nonerythropoietic tissues, TNF-α-dependent upregulation of EpoR has been described to mediate the tissue-protective action of Epo (Brines & Cerami, 2008). To investigate whether this also applies for bladder epithelial cells, we exposed cells to bacterial stimuli, E. coli NU14, and determined the mRNA expression of EpoR at different time points after stimulation. The expression of EpoR was induced in a bimodal

manner, with a first peak at three (5637 cells) or 6 h (primary cells) and a second upregulation after 24 h of stimulation (Fig. 2a). This first peak was very low in T24 cells stimulated with bacteria alone. When, however, these cells were costimulated with ARA290, EpoR expression was upregulated 3 h after costimulation (P<0.05; Fig. 2b). Enhanced and earlier EpoR upregulation in the presence of ARA290 was also observed for 5637 and primary bladder epithelial cells, although the effect was less pronounced (data not shown). In the monocytic cell line THP-1, a similar pattern was observed, but expression peaked earlier, after 1 and 12 h of stimulation, respectively (Fig. 2a). Additional stimulation with ARA290 showed no obvious additive effect.