Identification of the dopamine transporter SLC6A3 as a biomarker for patients with renal cell carcinoma

Western blot

Western Blot analyses were performed to determine the expression of six differentially expressed mRNAs on the protein level. We investigated eight corresponding ccRCC and normal renal tissues; the fresh-frozen tissue samples were located adjacent to the tissue used for RNA isolation. Approximately 50 mg tissue was homogenized in a Precellys 24 (Peqlab, Erlangen, Germany) with 400 ?l Cell Lysis Buffer (Cell Signaling, Cambridge, United Kingdom) including protease inhibitor (Complete Mini EDTA-free, Roche, Basel, Switzerland). After determination of the protein concentration (BCA Protein Assay Kit, Pierce Biotechnology, Rockford, IL, USA), 30 ng protein was loaded per lane into a NuPAGE 3–8 % or 4–12 % denaturating PAA Gel (Life Technologies, Carlsbad, CA, USA) and separated in a XCell4 SureLock electrophoresis system (Life Technologies). A biotinylated protein ladder (catalog number 7727S, Cell Signaling) was used as molecular weight marker. After the transfer on 0.2 ?m nitrocellulose (XCell II, Life Technologies) and a 5 % milk powder (Merck, Darmstadt, Germany) protein block, the immunostaining procedure was performed with antibodies against SLC6A3 (#LS-B7715, LSBio, Seattle, WA, USA), SLC12A1 (#NBP1-80993, Novus Bio, Cambridge, United Kingdom), FXYD4 (#NBP1-84489, Novus Bio), NPTX2 (#ab115528, Abcam, Cambridge, United Kingdom), KNG1 (#ab124737, Abcam), NDUF4AL2 (#ab190007, Abcam), beta-actin (#A5316, Sigma) and GAPDH (#2118, Cell Signaling). The detection was carried out with horseradish peroxidase conjugated to secondary antibodies (anti-mouse-POD, #170-6516, Biorad, Munich, Germany; anti-rabbit-POD, #170-6515, Biorad; anti-biotin-POD, #7075, Cell Signaling). The development of the chemiluminescent signal was done by SuperSignal West Femto (Pierce Technology) and documented by the LAS 3000 Image Reader (Fujifil, Tokyo, Japan).