Methods in molecular biology
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Normal cellular functioning is maintained by macromolecular machines that control both core and specialized molecular tasks. These machines are in large part multi-subunit protein complexes that undergo regulation at multiple levels, from expression of requisite components to a vast array of post-translational modifications (PTMs). ⋯ Here we provide a framework for systematically studying these PTMs in the context of global protein-protein interaction networks. This analytical framework allows insight into which functions specific PTMs tend to cluster in, and furthermore which complexes either single or multiple PTM signaling pathways converge on.
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Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (AONs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Multiple exon skipping utilizing a cocktail of AONs can theoretically treat 80-90% of patients with Duchenne muscular dystrophy (DMD). The success of multiple exon skipping by the systemic delivery of a cocktail of AONs called phosphorodiamidate morpholino oligomers (PMOs) in a DMD dog model has made a significant impact on the development of therapeutics for DMD, leading to clinical trials of PMO-based drugs. Here, we describe the systemic delivery of a cocktail of PMOs to skip multiple exons in dystrophic dogs and the evaluation of the efficacies and toxicity in vivo.
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Protein post-translational modification (PTM) is an essential cellular regulatory mechanism, and disruptions in PTM have been implicated in disease. PTMs are an active area of study in many fields, leading to a wealth of PTM information in the scientific literature. ⋯ This chapter describes the use of iPTMnet ( http://proteininformationresource.org/iPTMnet/ ), a resource that integrates PTM information from text mining, curated databases, and ontologies and provides visualization tools for exploring PTM networks, PTM crosstalk, and PTM conservation across species. We present several PTM-related queries and demonstrate how they can be addressed using iPTMnet.
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Allergic asthma, caused by inhaled allergens such as house dust mite or grass pollen, is characterized by reversible airway obstruction, associated with an eosinophilic inflammation of the airways, as well as airway hyper responsiveness and remodeling. The inhaled allergens trigger a type-2 inflammatory response with involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high production of immunoglobulin E (IgE) antibodies. Consequently, renewed allergen exposure results in a classic allergic response with a distinct early and late phase, both resulting in bronchoconstriction and shortness of breath. ⋯ Finally, mice are challenged by three intranasal allergen administrations. We will describe the protocols as well as the most important read-out parameters including measurement of invasive lung function measurements, serum immunoglobulin levels, isolation of broncho-alveolar lavage fluid (BALF), and preparation of cytospins. Moreover, we describe how to restimulate lung single cell suspensions, perform flow cytometry measurements to identify populations of relevant immune cells, and perform ELISAs and Luminex assays to measure the cytokine concentrations in BALF and lung tissue.
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Glycosylation of membrane proteins plays a crucial role in various physiological events, including intercellular recognition and intermolecular interactions on the cell surface (Gornik et al., Biochim Biophys Acta 1820:1318-1326, 2012). To study composition and function of N-glycans on membrane proteins one has to have an efficient and reproducible analytical method, which includes protein extraction and analysis of glycans. In this chapter we provide an analytical approach that includes cloud-point extraction (CPE) of total membrane proteins with the non-ionic detergent Triton X-114 and subsequent analysis of their N-glycans using hydrophilic interaction liquid chromatography (HILIC)-UPLC/HPLC. The protocol presented here can be used for parallel analysis of both membrane and intracellular proteins.