As discussed previously, only two publications are available to date for the characterization of MCT6 function, and inhibitor/substrate identification.3,4 Interestingly in these studies, MCT6-mediated bumetanide uptake was not inhibited by prototypical MCT 1C4 ligands, such as the short-chain monocarboxylic acids L-lactic acid and pyruvic acid, which agrees with our experimental data. NH). All enzymes were purchased from New England Biotechnology (Ipswich, MA). The gel extraction and PCR purification kits were purchased from Qiagen (Valencia, CA). DNA purity and concentration were verified using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Rockford, IL). A ZOE fluorescent cell imager made by Bio-Rad (Hercules, CA) was used for fluorescent microscopy. The mouse anti-Egfp antibody (JL-8, Cat. No. 632381) was purchased from Clontech (Mountain View, CA). Generation of the pGH19-hMCT6 Vector For these studies, we generated injectable cRNA encoding for the human MCT6 protein from the transcription of a MCT6 genetic construct. Briefly, TH287 total RNA derived from human kidney cortical tissue (obtained from Ohio State University Tissue Procurement Services, National Cancer Institute Collaborative Human Tissue Network (CHTN), Columbus, OH) was isolated using TRIzol Reagent according to the manufacturers instructions. After checking the concentration, purity, and TH287 stability using the FlashGel System, cDNA was generated by performing RT-PCR using the BioRad CFX Connect RT TH287 System. Using this cDNA library, sequence specific primers were designed to amplify the specific cDNA fragment encoding for the MCT6 gene (cells, and the ligated product was isolated, purified, and confirmed via sequencing. Preparation of cRNA and Verification of Protein Expression in Oocytes The cRNA encoding for MCT6 was transcribed from ovaries. The oocytes that were injected ranged from Dumont stages IVCVI, and approximately 13.8 nL of cRNA or water was injected into each Rabbit Polyclonal to EDG7 oocyte (~20 ng). The oocytes were then incubated in OR3 medium (which was prepared as described previously24) at 18 C for 3 to 4 4 days. All cRNA was checked for purity, concentration, stability, and correct size utilizing a NanoDrop 1000 and FlashGel System. Due to the inability of a variety of antibodies against human MCT6 to verify positive protein expression 3C4 days postinjection in our oocytes, our lab developed an MCT6 Egfp-tagged construct using similar methods as described previously in order to verify the protein expression and localization to the membrane using a fluorescent imager and an anti-Egfp antibody. Briefly, 3 days postinjection, oocytes were washed with 200 mOsm Tris-buffered saline (TBS) and visualized using a fluorescence microscope. In addition, Western blotting was performed from water-injected, MCT6 cRNA-injected, Egfp cRNA-injected, and MCT6-Egfp cRNA-injected oocytes from day 1 to day 4 postinjection. Briefly, 5 oocytes from each group each day were washed three times in 200 mOsm TBS and homogenized using 500 for 10 min. The soluble fraction was then spun in a Spin-X centrifuge tube filter (Corning Costar, 0.45 for 10 min. For each soluble fraction, 25 0.05. Data analysis was performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego CA). The MCT6-specific uptake rates of bumetanide were obtained from the uptake value at 30 min, which was previously shown to be in the linear range.3 The inhibition of bumetanide uptake by flavonoids was calculated by fitting with eq 1 using weighted nonlinear regression analysis (ADAPT 5; Biomedical Simulations Research, University of South California, Los Angeles, CA). is the percentage of uptake rate of bumetanide in the presence of flavonoids compared with the control, is the concentration of flavonoids, is the MCT6-mediated bumetanide uptake rate (pmol/oocyte/30 min), is the concentration of bumetanide (Oocytes.