Western blot analysis of sub-cellular fractionated samples using the Odyssey Infrared Imaging System

Misawa, Yukiko; Li, Ying; Rekosh, David; Hammarskjöld, Marie‐Louise

doi:10.1038/nprot.2006.219

Cited by 7 publications

(5 citation statements)

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“…Perform western blotting on cell lysates 43 .Incubate western blots overnight at 4 °C with primary antibodies specific to EphA2, phosphotyrosine and actin.Incubate western blots at room temperature for 1 h with isotype-matched secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 780.Image western blots with an Odyssey Infrared Scanning System (LI-COR Biotechnology).…”

Section: Methodsmentioning

confidence: 99%

Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling

et al. 2011

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physical inputs, both internal and external to a cell, can directly alter the spatial organization of cell surface receptors and their associated functions. Here we describe a protocol that combines solid-state nanolithography and supported lipid membrane techniques to trigger and manipulate specific receptors on the surface of living cells and to develop an understanding of the interplay between spatial organization and receptor function. While existing protein-patterning techniques are capable of presenting cells with well-defined clusters of protein, this protocol uniquely allows for the control of the spatial organization of laterally fluid receptor-ligand complex at an intermembrane junction. a combination of immunofluorescence and single-cell microscopy methods and complementary biochemical analyses are used to characterize receptor signaling pathways and cell functions. the protocol requires 2–5 d to complete depending on the parameters to be studied. In principle, this protocol is widely applicable to eukaryotic cells and herein is specifically developed to study the role of physical organization and translocation of the EphA2 receptor tyrosine kinase across a library of model breast cancer cell lines.

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Section: Methodsmentioning

confidence: 99%

Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling

et al. 2011

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“…Membranes were subsequently probed with a monoclonal β-actin antibody (Sigma, St. Louis, MO) followed by a sheep anti-mouse secondary antibody conjugated with IR-Dye800 (Rockland Immunochemicals, Philadelphia, PA). APOBEC3G expression is expressed as fluorescent intensity (Relative Light Units, RLU) of APOBEC3G bands divided by the fluorescent intensity (RLU) of the β-actin band [56] . For quantification of virion packaged APOBEC3G, virions were concentrated by centrifugation of culture supernatants through a 20% sucrose cushion at 125,000× g for 45 minutes and normalized for their p24 content with viral lysis buffer [50 mM Tris (pH 8.0), 40 mM KCl, 50 mM NaCl, 5 mM Na 2 EDTA, 10 mM DTT and 0.1% (v/v) Triton X-100].…”

Section: Methodsmentioning

confidence: 99%

Differences in APOBEC3G Expression in CD4+ T Helper Lymphocyte Subtypes Modulate HIV-1 Infectivity

et al. 2009

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The cytidine deaminases APOBEC3G and APOBEC3F exert anti–HIV-1 activity that is countered by the HIV-1 vif protein. Based on potential transcription factor binding sites in their putative promoters, we hypothesized that expression of APOBEC3G and APOBEC3F would vary with T helper lymphocyte differentiation. Naive CD4+ T lymphocytes were differentiated to T helper type 1 (Th1) and 2 (Th2) effector cells by expression of transcription factors Tbet and GATA3, respectively, as well as by cytokine polarization. APOBEC3G and APOBEC3F RNA levels, and APOBEC3G protein levels, were higher in Th1 than in Th2 cells. T cell receptor stimulation further increased APOBEC3G and APOBEC3F expression in Tbet- and control-transduced, but not in GATA3-transduced, cells. Neutralizing anti–interferon-γ antibodies reduced both basal and T cell receptor-stimulated APOBEC3G and APOBEC3F expression in Tbet- and control-transduced cells. HIV-1 produced from Th1 cells had more virion APOBEC3G, and decreased infectivity, compared to virions produced from Th2 cells. These differences between Th1- and Th2-produced virions were greater for viruses lacking functional vif, but also seen with vif-positive viruses. Over-expression of APOBEC3G in Th2 cells decreased the infectivity of virions produced from Th2 cells, and reduction of APOBEC3G in Th1 cells increased infectivity of virions produced from Th1 cells, consistent with a causal role for APOBEC3G in the infectivity difference. These results indicate that APOBEC3G and APOBEC3F levels vary physiologically during CD4+ T lymphocyte differentiation, that interferon-γ contributes to this modulation, and that this physiological regulation can cause changes in infectivity of progeny virions, even in the presence of HIV-1 vif.

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“…4b). To verify the efficiency of the fractionation, blots were probed with anti-tubulin, which has previously been used as a control for subcellular fractionations (Katsuno et al 2003;Misawa et al 2006). Tubulin was detected in non-nuclear (cytoplasmic) fractions, but not in nuclear fractions (Fig.…”

Section: Subcellular Localizationmentioning

confidence: 99%

Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in Dictyostelium discoideum

Huber

O’Day

2011

Histochem Cell Biol

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The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.

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Western blot analysis of sub-cellular fractionated samples using the Odyssey Infrared Imaging System

Cited by 7 publications

References 0 publications

Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling

Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling

Differences in APOBEC3G Expression in CD4+ T Helper Lymphocyte Subtypes Modulate HIV-1 Infectivity

Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in Dictyostelium discoideum

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