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Mol. Cancer 15, 32. ARHGEF15 overexpression worsens the prognosis in patients with pancreatic ductal adenocarcinoma through enhancing the motility and proliferative activity of the cancer cells. 2016

Fukushima, H., Yasumoto, M., Ogasawara, S., Akiba, J., Kitasato, Y., Nakayama, M., Naito, Y., Ishida, Y., Okabe, Y., Yasunaga, M., Horiuchi, H., Sakamoto, E., Itadani, H., Mizuarai, S., Oie, S. and Yano, H.

Notes: Hs766T cells were transfected with a purchased HaloTag®-ARHGEF15 fusion expression vector in pFN21A and siRNA expression plasmids directed to the ARHGEF15 or nonsense sequence using the ViaFect™ Transfection Reagent (no details provided)., ARHGEF15 expression was visualized but the specific HaloTag® Ligand was not specified. (4667)

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16, 37–47. The TIP60 complex is a conserved coactivator of HIF1A. 2016

Perez-Perri, J.I., Dengler, V.L., Audetat, K.A., Pandey, A., Bonner, E.A., Urh, M., Mendez, J., Daniels, D.L., Wappner, P., Galbraith M.D. and Espinosa, J.M.

Notes: HaloTag® Pull-Down Assay
HEK293T (12 × 106 cells) were plated and grown to 70–80% confluence (approximately 18 hours). The cells were then transfected (using FuGENE® HD Transfection Reagent [Cat.# E2311]) with either 30µg of HaloTag(HT)-HIF1A or HT-alone control vector (vectors available by custom order from Promega Custom Assay Services). Clarified lysates from both HT-HIF1A and HT-alone control cells were prepared and incubated with HaloLink™ Resin (HaloTag® Mammalian Pull-Down System [Cat.# G6500, G6504]). Proteins were digested with trypsin, and digestion was quenched with formic acid. Digested peptides were analyzed by mass spectrometry.

NanoBRET™ Assay
HCT116 and HEK293 cells (8 ×105) were plated in each well of a 6-well plate and co-transfected with one of three acceptors: HT-Pontin, HT-Reptin or HT-TIP60, in combination with the HIF1A-NanoLuc(NL) donor. The following NanoBRET pairs used are available by custom order from Promega Custom Assay Services: HIF1α-NLuc + HT-TIP60, HIF1α-NLuc + HT-Pontin or HIF1α-NLuc + HT-Reptin. (4718)

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ACS Med. Chem. Lett. 7(5), 531–6. Fragment-based discovery of a selective and cell-active benzodiazepinone CBP/EP300 bromodomain inhibitor (CPI-637). 2016

Taylor, A.M., Côté, A., Hewitt, M.C., Pastor, R., Leblanc, Y., Nasveschuk, C.G., Romero, F.A., Crawford, T.D., Cantone, N., Jayaram, H., Setser, J., Murray, J., Beresini, M.H., de Leon Boenig, G., Chen, Z., Conery, A.R., Cummings, R.T., Dakin, L.A., Flynn, E.M., Huang, O.W., Kaufman, S., Keller, P.J., Kiefer, J.R., Lai, T., Li, Y., Liao, J., Liu, W., Lu, H., Pardo, E., Tsui, V., Wang, J., Wang, Y., Xu, Z., Yan, F., Yu, D., Zawadzke, L., Zhu, X., Zhu, X., Sims, R.J., 3rd, Cochran, A.G., Bellon, S., Audia, J.E., Magnuson, S. and Albrecht, B.K.

Notes: Researchers set out to identify CBP/EP300 bromodomain inhibitors potent to both in vitro targets and targets in cellular target engagement assays. They developed a series of selective probes of CBP/EP300 bromodomains identified first by fragment screening. They next substituted and modified parts of the fragments to improve potency and selectivity.

To determine whether improvements in CBP bromodomain inhibition that were observed in their biochemical assay would translate to a cellular context, they used a bioluminescence resonance energy transfer assay, NanoBRET, where a small CBP/EP300 bromodomain inhibitor disrupted the interaction between a HaloTag-labeled histone and bromodomain conjugated to NanoLuc® Luciferase.

NanoBRET™ CBP/Histone H3.3 Interaction Assay (Cat.# N1850) and FuGENE® HD Transfection Reagent (Cat.# E2311) were used in the cell-based assay. (4717)

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J. Biol. Chem. 290, 15030-41. Assembly of the Elongin A ubiquitin ligase is regulated by genotoxic and other stresses. 2015

Weems, J.C., Slaughter, B.D., Unruh, J.R., Hall, S.M., McLaird, M.B., Gilmore, J.M., Washburn, M.P., Florens, L., Yasukawa, T., Aso, T., Conaway, J.W. and Conaway, R.C.

Notes: In order to investigate the interaction of the Cullin-RING E3 ubiquitin ligase with Elongin A due to toxic damage to DNA, the authors chose a FRET assay consisting of mCherry-labeled Cullin-RING3 and HaloTag-labeled Elongin A (clone obtained from Promega as pFN21-TCEB3 then subcloned into another expression vector). Interaction of the two proteins was examined in UV irradiated HeLa and U2OS cells. As a control, a HaloTag Protein expression vector alone was used. Intracellular HaloTag-labeled protein visualization was accomplished with the HaloTag® R110Direct™ Ligand. Both cell lines were transfected at 50-60% confluency with 700ng of plasmid in glass bottom 35mm dishes. FuGENE® HD Reagent was used for HeLa cells and ViaFect™ Reagent was used for U2OS cells. (4674)

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J. Med. Chem. 58, 2718–36. 9H-Purine Scaffold Reveals Induced-Fit Pocket Plasticity of the BRD9 Bromodomain. 2015

Picaud, S., Strocchia, M., Terracciano, S., Lauro, G., Mendez, J., Daniels, D.L., Riccio, R., Bifulco, G., Bruno, I. and Filippakopoulos, P.

Notes: The authors used bioluminescence resonance energy transfer (BRET) to test the ability of a bromodomain 9 ligand to disrupt binding to histone. HEK 293 cells were cotransfected with a histone H3.3-HaloTag® fusion vector and either NanoLuc®-BRD9 bromodomain or NanoLuc®-full-length BRD4 fusion vector. After 24 hours, the transfected cells were trypsinized, diluted in phenol red-free DMEM with or without 10nM of HaloTag® NanoBRET™ 618 Ligand and dispensed into a 96-well plate. One of two potential BRD-disrupting compounds, 7d or 11, was adding to a final concentration of 0.005–33μM, cells were incubated for 18 hours and NanoBRET™ Nano-Glo® Substrate (final concentration 10µM) was added. Fluorescence was measured and a corrected BRET ratio calculated. Cytotoxicity was assessed after the NanoBRET™ assay by incubating the cells with the CellTiter-Glo® Reagent for 30 minutes and measuring luminescence. To examine histone H3.3 localization, HEK 293 cells were transfected with the histone H3.3-HaloTag® fusion vector using FuGENE® HD Transfection Reagent. After 24 hours, cells were labeled with 5μM HaloTag® TMR ligand for 15 minutes before washing with complete medium, incubated for 30 minutes and imaged with a confocal microscope. (4568)

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Angewandte Chemie International Edition 54, 6217–21. LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor 2015

Clark, P.G.K., Vieira, L.C.C., Tallant, C., Fedorov, O., Singleton, D.C., Rogers, C.M., Monteiro, O.P., Bennett, J.M., Baronio, R., Müller, S., Daniels, D.L., Méndez, J., Knapp, S., Brennan, P.E. and Dixon, D.J.

Notes: To characterize the effectiveness of LP99, a potential bromodomain inhibitor, BRD7 and BRD9 were fused with NanoLuc® luciferase and histones H3.3 and H4 were fused with HaloTag® protein for use in BRET. The two proteins were expressed in HEK 293 cells, and the histone-HaloTag® fusions were fluorescently labeled with the HaloTag® NanoBRET™ 618 Ligand. Once the NanoBRET™ Nano-Glo® Substrate was added, NanoBRET™ ratios were assessed in the presence of varying concentrations of LP99. (4567)

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J. Immunol. Methods 426, 95–103. On-bead antibody-small molecule conjugation using high-capacity magnetic beads. 2015

Nath, N., Godat, B., Benink, H. and Urh, M.

Notes: This paper describes an improved method for on-bead conjugation of antibodies that overcomes the limitations of solution-based protocols (requirement for purified, high-concentration antibodies and the need for multiple buffer changes). The method uses HaloTag technology to immobilize protein A and G onto high-capacity magnetic beads. Antibodies are then captured and labeled on-bead. The authors demonstrate that the method is compatible with amine- and thiol-based chemistries as well as with  mouse and human antibody isotypes, both purified and in cell media. Protein G and Protein A-HaloTag fusion proteins were constructed by cloning the coding sequences for the Fc-binding domains between N-terminal HQ and C-teminal HaloTag sequences. To create Protein A and G beads, the purified Protein G-HaloTag and Protein A-HaloTag constructs were covalently attached to Magne HaloTag Beads--magnetic cellulose beads activated with HaloTag ligands. (4586)

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ACS Chemical Biology 10, 1797–1804. NanoBRET—A Novel BRET Platform for the Analysis of Protein–Protein Interactions. 2015

Machleidt, T, Woodroofe, C.C., Schwinn, M.K., Méndez, J.,  Robers, M.B., Zimmerman, K., Otto, P., Daniels, D.L., Kirkland, T.A., and Wood, K.V.

Notes: This paper introduces NanoBRET technology, which provides an improved alternative to conventional BRET protein interaction assays. NanoBRET assays combine the extremely bright NanoLuc luciferase with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The greater light intensity and improved spectral resolution of the NanoBRET assay results in increased detection sensitivity and dynamic range over current BRET technologies. Performance of the assay is demonstrated using several model systems, and the ability to image BRET in individual cells is illustrated. The  authors also demonstrate the application of NanoBRET in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells. (4575)

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ACS Chemical Biology 10, 2316-24. Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag. 2015

Ohana, R,F., Kirkland, T.A., Woodroofe, C.C., Levin, S., Uyeda, H.T., Otto, P., Hurst, R., Robers, M.B., Zimmerman, K., Encell, L.P., and Wood, K.V.

Notes: This paper describes a new method for capturing and characterizing the cellular targets of  small molecules identified during phenotypic screening of compound libraries. The method uses a novel chloroalkane tag that has minimal effect on compound potency or cell permeability, allowing capture of protein targets in living cells. Interacting proteins are captured onto immobilized HaloTag® protein. The rapid, selective nature of this process enables capture of low-affinity or low-abundance targets. Exchanging the chloroalkane tag for a fluorophore, putative targets identified by mass spectrometry can be verified for direct binding to the compound using resonance energy transfer. The authors used the interaction between histone deacetylases (HDACs) and the inhibitor Vorinostat (SAHA) as a model system, and were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets. (4578)

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J. Cell Sci. 127, 5261-72. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. 2014

Nakamura, T., Yoshitomi, Y., Sakai, K., Patel, V., Fukumoto, S. and Yamada, Y.

Notes: Epiprofin (Epfn) was expressed in HaCaT cells from various HaloTag® Fusion vectors with either full-length or deletion mutants of the CMV promoter to provide different levels of Epfn expression. (The authors do not state whether the N-terminal or C-terminal HaloTag® Fusion vectors were used). HaCaT 72hr proliferation was highest when using the CMVd3 promoter (e.g., pFC17A or pFN24A) for the lowest expression level. Reporter assays were performed on in the HaCaT cells as well and were measured with the Dual-Luciferase® Reporter Assay System on a GloMax® Instrument. Transfection studies performed with primary keratinocytes utilized the ViaFect™ Transfection Reagent using the reverse transfection method. (4690)

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Proc. Natl. Acad. Sci. USA 111(38), 13990–5. Tyrosine phosphorylation of GluK2 up-regulates kainate receptor-mediated responses and downstream signaling after brain ischemia 2014

Zhu, Q.J., Kong, F.S., Xu, H., Wang, Y., Du, C.P., Sun, C.C., Liu, Y., Li, T. and Hou, X.Y.

Notes: In this study the authors looked for molecular mechanisms underlying the role of kainite receptors in ischemic stroke. In their studies, the researchers examined binding of Src kinase to GluK2, and the site of this interaction. A GST pulldown assay confirmed a direct interaction between GluK2 and Src in vitro. Then a bioluminescence resonance energy transfer (BRET) assay was used to examine this GluK2-Src interaction in live HEK293 cells, using NanoLuc® Luciferase as the energy donor, and HaloTag-labeled GluK2 as the energy acceptor. As reported, the co-expression of NLuc and HaloTag® fusions resulted in a significant NanoBRET ratio. The authors added untagged GluK2 as a competitor of the GluK2-HaloTag and Src-NLuc interaction, which resulted in reduction of the NanoBRET ratio. These results demonstrated that GluK2 interacts directly with Src in living cells. The GloMax® Discover Detection System was used to measure the output of these assays.

Their source of NanoLuc® Luciferase and HaloTag® tags was the NanoBRET™ PPI Starter System (Cat.# N1811, N1821). (4696)

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Proc. Natl. Acad. Sci. USA 112, 148–153. Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling. 2014

Wang, J., Ren, J., Wu, B., Feng, S., Cai, G., Tuluc, F., Peränen, J. and Guo, W.

Notes: To investigate whether protein conformation of Rabin8 plays a role in autoinhibition, the authors created a Rabin8 fusion construct with NanoLuc® luciferase at the N terminus and HaloTag® protein at the C terminus so that they can use BRET as an indication of protein conformation. A t-SRARE protein, syntaxin-4 (STX4), which is known to have a closed conformation, was constructed with the same NanoLuc® luciferase-STX4-HaloTag® protein configuration for use as a positive control. Both the control STX4 protein and Rabin8 were expressed in E. coli, the NanoBRET™ Nano-Glo® Substrate added and fluorescence measured. As a negative control, TEV protease was used to cleave the HaloTag® sequence from the protein fusions, eliminating the NanoBRET™ signal. NanoBRET™ signals were determined from experiments comparing Rabin8 with a gain-of-function Rabin8 mutant, exposing Rabin8 to constitutively active ERK2 or a kinase-dead ERK2 and assessing wildtype Rabin8 versus Rabin8-4D, where the aspartates acted as phosphorylation mimics. (4566)

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ACS Med. Chem. Lett. 5, 1190–1195. 1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain. 2014


Demont, E.H., Bamborough,P., Chung,C., Craggs, P.D., Fallon, D., Gordon, L.J., Grandi, P., Hobbs, C.I., Hussain, J., Jones, E.J., Le  A., Michon, A., Mitchell, D.J., Prinjha, R.K., Roberts, A.D., Sheppard, R.J, and Watson, R.J.

Notes: In this paper the authors report on the discovery, binding mode, and structure:activity relationship of the first potent, selective series of inhibitors of the BRPF1 (bromodomain and PHD finger-containing)  bromodomain.  Bromodomains are specific protein modules present in a group of chromatin-regulator proteins responsible for “reading” acetylated lysine residues. Although some bromodomain-containing proteins (BCPs), such as those in the BET subfamily, are well characterized and have been identified as potential therapeutic targets, other BCPs, including those in the BPRF subfamily, are less well understood.  These authors set out to generate selective inhibitors of the BRPF1 domain in order to better understand the functional role of this specific bromodomain region. Using an inhibitor discovery strategy based on other known compound-bromodomain interactions, a potent, selective inhibitor of the BRPF1 bromodomain was identified, synthesized, and characterized using in vitro methods.  To demonstrate the function of this compound in live cells, the NanoBRET™ assay for protein:protein interactions (PPI) was used. The NanoBRET™ PPI assay enabled the authors to demonstrate both the cell permeability of the newly identified compound and also the ability of the compound to disrupt chromatin binding of the BRPF1 domain. NanoLuc® Luciferase-tagged BRPF1 bromodomain and HaloTag®-labeled Histone H3.3 were used for the NanoBRET™ assay in HEK293 cells. Dose-response curves performed with the NanoBRET™ assay enabled calculation of the cellular IC50 of the newly identified compound. A less active control analog compound was unable to inhibit the BRPF1 bromodomain:Histone H3.3 interaction, demonstrating assay specificity. Finally, the newly identified compound was inactive in NanoBRET™ assays using a second BRPF1 isoform containing a natural insertion, a result that was consistent with the proposed compound mode of action. Confirmation that the new identified compound can enter cells and disrupt the BRPF1 bromodomain:chromatin interaction in a cellular environment suggests that it may be a useful compound for studying the physiological role and therapeutic potential of BCPs containing the BRPF1 bromodomain. (4514)

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Protein Expr. Purif. 89, 62–72. Application of HaloTag technology to expression and purification of cannabinoid receptor CB2. 2013

Locatelli-Hoops, S., Sheen, Fangmin C., Zoubak, L., Gawrisch, K. and Yeliseev, A.A.

Notes: The HaloTag® Protein Tag was used to tag, immobilize and purify functional cannabinoid receptor CB2, a GPCR,  after induction of expression in E. coli.  (4263)

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Cell 154, 541–555. KDM4A Lysine Demethylase Induces Site-Specific Copy Gain and Rereplication of Regions Amplified in Tumors 2013

Black, J.C., Manning, A.L., Van Rechem, C., Kim, J., Ladd, B., Cho, J., Pineda, C.M., Murphy, N., Daniels, D.L., Montagna, C., Lewis, P.W., Glass, K., Allis, C.D., Dyson, N.J., Getz, G. and Whetstine, J.R.

Notes: The HaloTag® protein tag was used in experiments to identify protein partners of KDM4A that involved in site-specific copy number gain in tumors, specifically at the 1q12h region. Expression constructs were transfected into HEK293T cells using the FuGENE® HD Transfection Reagent. The HaloTag-KDM4A (Cat.# FHC00602) and HaloTag-Suv39h1 (Cat.# FHC09879) expression constructs were obtained from the Kazusa DNA Research Institute (Kisarazu, Japan). Interacting proteins identified included DNA polymerase subunits and members of the minichromosome maintenance (MCM) complex. (4408)

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EMBO J. 32, 645–55. TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS. 2013

Deplus, R., Delatte, B., Schwinn, M.K., Defrance, M., Méndez, J., Murphy, N., Dawson, M.A., Volkmar, M., Putmans, P., Calonne, E., Shih, A.H., Levine, R.L., Bernard, O., Mercher, T., Solary, E., Urh, M. Daniels, D. and Fuks, F.

Notes: These authors set out to determine how TET2 and TET3 proteins are involved in epigenetic regulation. To characterize proteins that interact with TET, the authors expressed full-length TET1, TET2 and TET3 as HaloTag® fusion proteins and performed protein pull-downs. They identified novel interactions between all three TET proteins and O-GlcNAc transferase (OGT), which catalyzes the addition of N-acetylglucosamine (GlcNAc) to numerous transcription factors, regulatory proteins and histones to activate or inhibit the target protein or recruit additional proteins. In this paper, they focused on TET2 and TET3, which showed the strongest interaction with OGT. They mapped TET2, TET3 and OGT binding throughout  the genome by expressing these proteins as HaloTag® fusion proteins in HEK293T cells, crosslinking the proteins and DNA, then capturing the fusion proteins and associated DNA fragments and performing high-throughput sequencing to show that TET2/3 and OGT colocalize at active gene promoters and were tightly clustered near transcription start sites.

For expression of HaloTag® fusion proteins and controls, HEK-293 cells were plated at 12 ×106 cells in a 150mm dish and grown to 70–80% confluency before transfection with 30µg of plasmid using the FuGENE® HD Transfection Reagent.

To assess whether TET2/3-OGT activity affects the interaction of SET1/COMPASS with chromatin, the authors used bioluminescence resonance energy transfer (BRET). They created a fusion protein consisting of the H3K4 methyltransferase SETD1A and NanoLuc® luciferase as the energy donor and a fluorescently labeled histone H3.3-HaloTag® fusion protein as the energy acceptor.  These BRET experiments confirmed that TET2/3-OGT activity is necessary for SET1/COMPASS complex function and showed that TET and OGT activities promote binding of SETD1A, a component of the SET1/COMPASS complex, to chromatin. This binding increases H3K4me3 levels. Thus, the authors’ data support a TET2/3-OGT-mediated mechanism for regulating the SET1/COMPASS complex and thus H3K4me3. (4262)

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Cell 153, 1327-1339. HIF1A Employs CDK8-Mediator to Stimulate RNAPII Elongation in Response to Hypoxia. 2013

Galbraith, M., Allen, M., Bensard, C., Wang, X., Schwinn, M., Qin, B., Long, H., Daniels, D., Hahn, W., Dowell, R., and Espinosa, J.

Notes: These authors identified a previously unknown interaction between the transcription factor HIF1A and the cyclin-dependent kinase CDK8 (a component of the Mediator complex) in the regulation of genes associated with cellular survival under low-oxygen conditions. As part of the study, HaloTag technology was used to identify proteins interacting with CDK8 in a colorectal cancer cell line. Specifically, cells were transfected with CDK8 and CDK19 HaloTag fusion constructs obtained from Kazusa Institute. The cell lysates were then used in pulldown assays to capture interacting proteins. The results showed that CDK8 and CDK19 are present in mutually exclusive Mediator complexes. Details of the transfection are as follows: HCT116 cells were plated in 150 mm dishes and grown to 70%–80% confluence before transfection with 30 μg of plasmid DNA using FuGENE HD Transfection Reagent. (4355)

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J. Bacteriol. 194, 1389-1400. Legionella pneumophila LidA affects nucleotide binding and activity of the host GTPase Rab1. 2012

Neunuebel, M.R., Mohammadi, S., Jarnik, M., and Machner, M.P.

Notes: In this study, the L. pneumophila protein Lem3 was expressed as a HaloTag® fusion protein and purified using HaloLink™ Resin. Lem3 was first cloned into the pFN22K HaloTag® Vector and the resultant HaloTag-Lem3 protein was expressed in Single-Step (KRX) competent cells before purification using the HaloTag® Protein Purification System. Lem3 was cleaved from the HaloLink™ Resin using TEV protease.

  (4350)

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Current Chemical Genomics 6, 72-78. HaloTag, a Platform Technology for Protein Analysis. 2012

Urh, M., and Rosenberg, M.

Notes: This paper provides an overview of the many applications of HaloTag® Technology. The authors describe the development of the technology, focusing on it's multifunctional utility for protein imaging, protein isolation and display, and in the study of protein complexes and interactions. They also discuss it's potential to facilitate proteomics research studies across complex biological systems at the biochemical, cell-based and whole animal level. (4325)

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Sci. Signal. 4(180), online. Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response 2011

Yuanzheng, H., Xu, Y., Zhang, C., Gao, X., Dykema, K.J., martin, K.R., Ke, J., Hudson, E.A., Khoo, S.K., Resau, J.H., Alberts, A.S., Mackeigan, J.P., Furge, K.A. and Xu, H.E.

Notes: Yuangheng He and colleagues asked how the weak alkaline compound chloroquine (CQ) enhances the anti-inflammatory effects of synthetic glucocorticoids like dexamethasone, which are used to treat a host of inflammatory and autoimmune diseases. In the process they explored the intersection of lysosomal degradation pathways and glucocorticoid receptor signaling. They used Dual-Glo® Luciferase Assay System to look at glucocorticoid receptor-mediated (GR) activation and repression of reporters in AD293 cells under a variety of conditions (presence or absence of CQ; stripped serum, loss of lysosome synthesis, inhibition of V-ATPase, etc). They also used HaloTag® protein fusions to observe the fate of GR populations in the presence or absence of CQ and in the presence or absence of compounds that impair proteasome function. Live-cell imaging of GR-HaloTag® protein fusions revealed a dynamic association of the GR with lysosomes. The authors showed that glucocorticoid signaling is regulated by lysosomes. (4203)

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Biochem. J. 436, 387–397. The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response. 2011

Maruyama, A., Nishikawa, K., Kawatani, Y., Mimura, J., Hosoya, T., Harada, N., Yamamato, M. and Itoh, K.

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag® CMV Flexi® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase® Reporter Assay System. (4123)

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Nat Chem. Biol. July 3, Epub. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. 2011

Neklesa, T.K., Tae, H.S., Schneekloth, A.R., Stulberg, M.J., Corson, T.W., Sundberg, T.B., Raina, K., Holley, S.A., and Crews, C.M.

Notes: These authors investigated whether HaloTag® technology could be used to specifically target and degrade intracellular proteins. They developed a method to degrade specific proteins of interest using a small molecule by tagging the HaloTag® protein with an adamantyl moiety. The hypothesis was that appending a hydrophobic residue to a protein surface domain would mimic partial denaturation and induce proteasomal degradation. They first used a HaloTag®-luciferase fusion protein to determine the biological activity of various small molecules that enhanced hydrophobicity. After selecting the small molecule that reduced luciferase activity in HEK293 cells, they went on to test their hypothesis in various in vitro and in vivo models. They were able to demonstrate degradation of various HaloTag®-linked transmembrane proteins expressed in HEK293 cells, to show degradations of target proteins in Zebrafish, and to show degradation of the HRAS oncogene product both in NIH3T3 cells and in a mouse tumor model. (4125)

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Protein Expr. Purif. 76, 154-164. HaloTag-based purification of functional human kinases from mammalian cells. 2010

Ohana, R.F., Hurst, R., Vidugiriene, J., Slater, M.R., Wood, K.V. and Urh, M.

Notes: The authors of this paper demonstrate the utility of the HaloTag® protein purification system for purifying functional proteins from mammalian cells. To this end five kinases were cloned into HaloTag® vectors, expressed in and purified from HEK293T cells. To demonstrate functionality of the purified recombinant kinases, activity was measured using the appropriate ADP-Glo™ Assay Kinase Enzyme Systems. (4145)

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Development 137, 901–11. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. 2010

Hattori, T., Müller, C., Gebhard, S., Bauer, E., Pausch, F., Schlund, B., Bösl, M.R., Hess, A., Surmann-Schmitt, C., von der Mark, H., de Crombrugghe, B. and von der Mark, K.

Notes: To study the role of the transcription factor Sox9 in the transition from cartilage to bone in newborn mice, the authors performed chromatin immunoprecipitation using the HaloCHIP™ System. Primary rib chondrocytes were transfected with a vector expressing full-length Sox9 with a HaloTag® protein tag, then proteins and DNA were cross-linked. DNA was isolated and sonicated, and the Sox9:DNA complexes were precipitated using the HaloLink™ Resin. The precipitated DNA then was amplified by PCR to determine that SOX9 binds to the SRY sites in the Vegfa gene. (4054)

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Methods in Mol. Biol. 577, 121-131. Pulse-Chase Experiment for the Analysis of Protein Stability in Cultured Mammalian Cells by Covalent Fluorescent Labeling of Fusion Proteins 2009

Kei Yamaguchi, Shinichi Inoue, Osama Ohara and Takahiro Nagase

Notes: The authors used the Halotag® Labeling Technology in pulse-chase experiments. They pulse labeled proteins in cultured mammalian cells. Using the HaloTag® Technology, they were able to monitor the degradation of the labeled protein, Smad1, that was induced by coexpression of Smurf1. They conclude that the HaloTag® Technology could be used to monitor the regulation of SMAD1 degradation. (4055)

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