Methods in molecular biology
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Higher-grade gliomas are distinguished by increased vascular endothelial cell proliferation and peritumoral edema. These are thought to be instigated by vascular endothelial growth factor, which in turn is regulated by cellular oxygen tension. Hypoxia inducible factor-1alpha (HIF-1alpha) is a main responder to intracellular hypoxia and is overexpressed in many human cancers, including gliomas. Here we present methods for investigating the role of HIF-1alpha in glioma growth in vivo and in vitro using RNA interference in U251, U87, and U373 glioma cells.
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Spinal cord injury (SCI) is a major public health problem with no known effective treatment. Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult leading to neuronal dysfunction and death; yet, the molecular mechanisms underlying its dysfunction are poorly understood. Furthermore, while use of imaging methods (e.g., computed tomography scans and magnetic resonance imaging) may help define injury severity and location, they do not elucidate biological mechanisms of SCI progression. ⋯ Spinal cord contusion is an extensively used SCI model in rats that best represents the etiology of SCI in humans. In this chapter, we describe a two-dimensional (2D) gel electrophoresis-based proteomic approach to investigate the injury-related differences in the proteome and phosphoproteome of spinal cord lesion epicenter at 24 h after spinal cord contusion in rats. The purpose of this study is to elucidate the mechanisms of acute spinal cord dysfunction, as well as discover novel biomarker candidates to evaluate the biological mechanisms of SCI progression and the injury severity.
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With the appearance of a new generation of high-performance hybrid mass spectrometers, high accuracy (sub-parts-per-million) mass spectrometry is becoming increasingly available to a wider scientific community. Here we discuss the advantages of such mass spectrometric instrumentation in the global analysis of protein phosphorylation. We describe a detailed workflow for fractionation and enrichment of phosphopeptides from digests of whole cell/tissue lysates by strong cation exchange and TiO(2) chromatography and their subsequent measurement on an LTQ-Orbitrap mass spectrometer under several acquisition regimes.
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RNAi holds promise for neurodegenerative disorders caused by gain-of-function mutations. We and others have demonstrated proof-of-principle for viral-mediated RNAi in a mouse model of motor neuron disease. ⋯ This chapter describes the design, production, and titration of lentivirus and adeno-associated virus capable of mediating SOD1 knockdown in vivo. The delivery of the virus to the spinal cord directly, through intraspinal injection, or indirectly, through intramuscular injection, is also described, as well as the methods pertaining to the analysis of spinal cord transduction, SOD1 silencing, and determination of motor neuron protection.