Figure 4. Schematic representation for the regulation of GLCE expression in normal breast tissues and breast tumors. sonicated and centrifuged for 10 min at 12,000 g. The protein concentration was quantified using Quant-iT Protein Assay Kit (Invitrogen). Total proteins (30 μg) were treated in NuPAGE LDS Sample Buffer (Invitrogen) with 10% β-mercaptoethanol for 5 min at 100°C, resolved in 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% milk for 1 h and incubated with primary antibodies (rabbit anti- GLCE polyclonal serum (GenScript Corporation), 1:2,000) overnight at 4°C and secondary peroxidase-conjugated antibody [polyclonal goat anti-rabbit (Pierce)] for 1 h at RT. Proteins were Acknowledgments The work was supported by the research grants from Russian Foundation for Basic Research (11-04-90400-Ukr_f_a); Ukranian State Foundation of Fundamental Research (F40/146- 2011, F46/457-2011); Swedish Institute (Visby Programme 2011/00888); UICC International Cancer Technology Transfer Fellowship (TYP, ICRETT-09-069); FEBS Short-term Fellowship (TYP); Karolinska Institute; ERZ was supported by the Swedish Cancer Society and Swedish Research Council. detected with an Opti-4CN Substrate Kit (Bio- Rad) according to the manufacturer’s instructions. The gels were scanned using the “DNA Analyzer” system (Vilber Lourmat) and GLCE protein lev- els were estimated from the intensity of GLCE fragment normalized against the intensity of GAPDH using the TotalLab program (Nonlinear Dynamics). Statistical analysis. Pearson’s correlation was used to determine the association between miRNA-218 and GLCE expression. 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