Pulse-Chase Experiment for the Analysis of Protein Stability in Cultured Mammalian Cells by Covalent Fluorescent Labeling of Fusion Proteins

Laboratory of Human Gene Research, Department of Human Genome Research, Kazusa DNA Research Institute, Chiba, Japan.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 02/2009; 577:121-31. DOI: 10.1007/978-1-60761-232-2_10
Source: PubMed


We used HaloTag labeling technology for the pulse labeling of proteins in cultured mammalian cells. HaloTag technology allows a HaloTag-fusion protein to covalently bind to a specific small molecule fluorescent ligand. Thus specifically labeled HaloTag-fusion proteins can be chased in cells and observed in vitro after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Fluorescent HaloTag ligand allows quantification of the labeled proteins by fluorescent image analysis. Herein, we demonstrated that the method allows analysis of the intracellular protein stability as regulated by protein-degradation signals or an exogenously expressed E3 ubiquitin ligase.

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    • "However, as seen for the total cell lysate (Figures 1A and 1B), the decrease in cytosolic TIAM1 was partially reversed, whereas the depletion of the membrane fraction was sustained through all subsequent time points (Figures 1E and 1F). To further examine the decrease in TIAM1 seen within the first hour of HGF stimulation , we engineered MDCKII cells to inducibly express Halotagged TIAM1 and analyzed the downregulation of TIAM1-Halo at cell-cell adhesions as well as in the cytoplasm using a pulse-chase method described elsewhere (Yamaguchi et al., 2009). We detected significant TIAM1 depletion from cell-cell adhesions but less so from the cytoplasm of HGF-stimulated cells (Figures 1G–1I). "
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    ABSTRACT: The E3 ubiquitin ligase HUWE1, deregulated in carcinoma, has been implicated in tumor formation. Here, we uncover a role for HUWE1 in cell migration and invasion through degrading the RAC activator TIAM1, implying an additional function in malignant progression. In MDCKII cells in response to HGF, HUWE1 catalyzes TIAM1 ubiquitylation and degradation predominantly at cell-cell adhesions, facilitating junction disassembly, migration, and invasion. Depleting HUWE1 or mutating the TIAM1 ubiquitylation site prevents TIAM1 degradation, antagonizing scattering, and invasion. Moreover, simultaneous depletion of TIAM1 restores migration and invasion in HUWE1-depleted cells. Significantly, we show that HUWE1 stimulates human lung cancer cell invasion through regulating TIAM1 stability. Finally, we demonstrate that HUWE1 and TIAM1 protein levels are inversely correlated in human lung carcinomas. Thus, we elucidate a critical role for HUWE1 in regulating epithelial cell-cell adhesion and provide additional evidence that ubiquitylation contributes to spatiotemporal control of RAC. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 12/2014; 10(1). DOI:10.1016/j.celrep.2014.12.012 · 8.36 Impact Factor
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    • "As a versatile tool, the HaloTag technology has attracted much attention for a broad array of biomedical applications such as in vitro optical imaging [1] [2] [3], in vivo cell labeling/imaging [4] [5], protein purification/trafficking [6] [7], study of protein-protein and protein-DNA interactions [8], analysis of protein stability [9], highthroughput assays [10], single molecule force spectroscopy [11], ribosome tagging [12], among many others [13]. The HaloTag technology involves two key components: the HaloTag protein and HaloTag ligands (HTLs). "
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    ABSTRACT: THE GOAL OF THIS STUDY IS TO EMPLOY THE HALOTAG TECHNOLOGY FOR POSITRON EMISSION TOMOGRAPHY (PET), WHICH INVOLVES TWO COMPONENTS: the HaloTag protein (a modified hydrolase which covalently binds to synthetic ligands) and HaloTag ligands (HTLs). 4T1 murine breast cancer cells were stably transfected to express HaloTag protein on the surface (termed as 4T1-HaloTag-ECS, ECS denotes extracellular surface). Two new HTLs were synthesized and termed NOTA-HTL2G-S and NOTA-HTL2G-L (2G indicates second generation, S stands for short, L stands for long, NOTA denotes 1,4,7-triazacyclononane-N,N'N''-triacetic acid). Microscopy studies confirmed surface expression of HaloTag in 4T1-HaloTag-ECS cells, which specifically bind NOTA-HTL2G-S/L. Uptake of (64)Cu-NOTA-HTL2G-L in 4T1-HaloTag-ECS tumors (4.3 ± 0.5, 4.1± 0.2, 4.0 ± 0.2, 2.3 ± 0.1, and 2.2 ± 0.1 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) was significantly higher than that in the 4T1 tumors (3.0 ± 0.3, 3.0± 0.1, 3.0 ± 0.2, 2.0 ± 0.4, and 2.4 ± 0.3 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) at early time points. In comparison, (64)Cu-NOTA-HTL2G-S did not demonstrate significant uptake in either 4T1-HaloTag-ECS or 4T1 tumors. Blocking studies and autoradiography of tumor lysates confirmed that (64)Cu-NOTA-HTL2G-L binds specifically to HaloTag protein in the 4T1-HaloTag-ECS tumors, corroborated by histology. HaloTag protein-specific targeting and PET imaging in vivo with (64)Cu-NOTA-HTL2G-L serves as a proof-of-principle for future non-invasive and sensitive tracking of HaloTag-transfected cells with PET, as well as many other studies of gene/protein/cell function in vivo.
    American Journal of Translational Research 05/2013; 5(3):291-302. · 3.40 Impact Factor
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    • "MIN6/insulin-HT stable cells were at a density of 2×106 cells in 6-cm plate. For labeling newly synthesized insulin-HT, the cells were first treated with blocking probe (10 µM HT-succinimidyl ester (O4) reagent) [16] for 1 hour to mask previously synthesized insulin-HT in the cell. After washing out excess blocking probe, the cells were incubated in modified Krebs-Ringer buffer (2 mM glucose (low glucose: LG), 120 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, 15 mM HEPES (pH 7.4), 0.1% bovine serum albumin) for 1 hour, and further incubated with HT probe (either 5 µM TMR or 1 µM HT-biotin for fluorescence and biochemical analysis, respectively) in buffer for 30 minutes. "
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    ABSTRACT: Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic β-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.
    PLoS ONE 10/2012; 7(10):e47921. DOI:10.1371/journal.pone.0047921 · 3.23 Impact Factor
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