Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.
Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre-and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.
Receptor-tyrosine kinases (RTKs) are transmembrane proteins that have been implicated in various cancers and are considered therapeutic targets. The phosphorylation of RTKs on tyrosine residues leads to their activation. Therefore, methods to identify the effect of RTK inhibitors or modifying antibodies on RTK phosphorylation are effective tools for drug screening. The Proteome Profiler 96 Human Phospho-RTK Antibody Array is a unique and powerful, plate-based multiplex immunoassay that utilizes ELISA techniques to measure changes in the phosphorylation of multiple RTKs simultaneously. In this assay, antibody/antigen reactions take place on the surface of a microplate that has been pre-spotted with antibodies against each RTK. Each spot corresponds to a unique analyte. A horseradish peroxidase-conjugated anti-Phospho-Tyrosine detection antibody is subsequently added to each well. A chemiluminescent substrate mix and a camera imaging system are used to determine the relative amount of analyte bound in individual spots. Experiments were performed by incubating the MDA-MB-453 breast cancer cell line with specific kinase inhibitors or with antibodies to ErbB receptors prior to NRG1- β1/HRG1- β1 treatment. Cells were lysed directly in 96-well plates and transferred to the Proteome Profiler 96 Human Phospho-RTK Array for analysis. NRG1- β1/HRG1- β1-dependent tyrosine phosphorylation of all four ErbB receptors was monitored simultaneously and the effects of different kinase inhibitors or modifying antibodies were determined. Proteome Profiler 96 Antibody Arrays offer the advantages of small sample and volume size requirements with as little as 5 μg of total cellular protein in a total volume of 100 μL being required, while still offering the specificity and sensitivity of sandwich immunoassays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5572.
Neutrophils are essential effector cells for mediating rapid host defense and their insufficiency arising from therapy-induced side-effects, termed neutropenia, can lead to immunodeficiency-associated complications. In autologous hematopoietic stem cell transplantation (HSCT), neutropenia is a complication that limits therapeutic efficacy. Here, we report the development and in vivo evaluation of an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel, with myeloid responsive degradation behavior. In mouse models of immune deficiency, we show that the infiltration of functional myeloid-lineage cells, specifically neutrophils, is essential to mediate HA cryogel degradation. Post-HSCT neutropenia in recipient mice delayed degradation of HA cryogels by up to 3 weeks. We harnessed the neutrophil-responsive degradation to sustain the release of granulocyte colony stimulating factor (G-CSF) from HA cryogels. Sustained release of G-CSF from HA cryogels enhanced post-HSCT neutrophil recovery, comparable to pegylated G-CSF, which, in turn, accelerated cryogel degradation. HA cryogels are a potential approach for enhancing neutrophils and concurrently assessing immune recovery in neutropenic hosts.
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