The Analytical Core will be organized to provide high quality, reproducible, low cost analytical services to the four sub-projects as well as the Animal Core. The Analytical core will be organized around three major services; 1) analytical methods common to all subprojects that need core services, 2) cell culture of human proximal tubular cells, and 3) imaging. Expert consultants and/or collaborators have been lined up to support each of these activities. The analytical methods service will provide Western blotting for the AT1, D1, and D3 receptors, and GRK4. Second messenger stimulation studies will also be performed including cAMP accumulation. We will also determine sodium transport by measuring NaKATPase and direct sodium flux using Sodium Green. Human, rat, and mouse tissue and/or blood will be genotyped using our automated genotyping system based on the Serologicals molecular beacon assay developed in our laboratory. The cell culture service will provide high quality cultured cells; including human proximal tubular cells will be cultured for the four subprojects in the analytical core to assure that the consistent cell clones and passages are used throughout the project. All human cells will be genotyped for SNPs related to hypertension described in projects by Felder and Carey. The imaging service will feature a state of the art confocal laser scanning upright microscope capable of imaging realtime events in proximal tubular cells grown on transwell membranes in order to maintain cell polarity. The imaging service will be assisted by Amassi Periasamy, Ph.D. (an expert in cellular imaging and fluorescence measurement), and Dorothy Shaeffer (an expert in cell cytoskelatal actin). The analytical core will be responsible for searching for the latest molecular and biochemical techniques applicable to the 3 subprojects and sharing this information with the PPG team.
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