CORE C: PROTEIN EXPRESSION AND CELL MORPHOLOGY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. service for the examination of protein expression using western analysis combined with a variety of morphological approaches to localize levels of expression in tissues with heterogeneous populations of cells. The Core has integrated western analysis and morphological studies such that quantitative protein expression and imaging needs from basic morphological to complex 3-dimensional volumetric measurements or electron microscopy will be performed in the same laboratory providing a seamless support mechanism for individual projects. The services to be provided by the Core include protein overexpression, protein expression analysis in tissues and cells, transfections of cells, cell culture, cell lysate preparations, immunoprecipitations, tissue preparations, western blots, gel electrophoresis, nanoLC and MALDl sample preparation. The Core will also act as an interface with the Nevada Proteomics Core for mass spectral analysis and analysis of MALDl data. Integrated within this support system with be a structural analysis of tissues and visualization of the sites of protein expression within tissues and isolated cells. Support will be provided in the areas of light microscopy (bright field, phase contrast and differential interference contrast microscopy) for the proposed histological studies on visceral tissues. The Core will also provide morphological expertise in the areas of immunofluorescence (regular fluorescence and confocal microscopy) for the examination of protein expression in heterogeneous tissues that contain several cellular phenotypes. The Core will troubleshoot the use of antibodies (i.e. fixation protocols, non-specific labeling) to optimize use and the detection of proteins. Digital reconstructions will be deconvolved and 3-dimensional volume reconstructions performed when needed The core will also provide support for the examination of eGFP expression in tissues and isolated cells prior to the sorting of smooth muscle cells from smMHCICreleGFP mice. The Core will also provide expertise in the ultrastructural analysis of muscles and isolated myofilaments. Several areas of expertise will be supported including conventional electron microscopy, negative staining of isolated myofilaments, cryoelectron microscopy and immunogold labeling of proteins. Ultrastructural analysis is necessary to determine the pathological changes that occur in smooth muscle cells during hypertrophy and changes in structure due to diseases like type II DM. Analysis of actin filaments using negative staining and immunogold labeling will be provided to determine the binding sites of myosin light chain kinase and phosphatase.