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Citations Search

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Mol. Cell. Proteomics 17(7), 1432–47. A structured workflow for mapping human Sin3 histone deacetylase complex interactions using Halo-MudPIT affinity-purification mass spectrometry. 2018

Banks, C.A.S., Thornton, J.L., Eubanks, C.G., Adams, M.K., Miah, S., Boanca, G., Liu, X., Katt, M.L., Parmely, T.J., Florens, L., Washburn, M.P.

Notes: A novel method for affinity purification mass spectrometry (AP-MS) is described using Magne® HaloTag® Beads. The Sin3 histone deacetylase complex subunits were tagged, and interactions with the remaining components were detected. Cellular lysates were incubated with Magne® HaloTag® Beads to enrich for tagged proteins. Together, an interaction network for the chromatin remodeling complex is described. (5100)

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Neuron 98, 977–91. Excitatory and Inhibitory Neurons Utilize Different Ca2+ Sensors and Sources to Regulate Spontaneous Release. 2018

Courtney, N.A., Briguglio, J.S., Bradberry, M.M., Greer, C., Chapman, E.R.

Notes: To localize and see Doc2 in live neurons the authors generated C-terminal HaloTag® fusion proteins of Doc2 isoforms, then conjugated these fusions with JF646 HaloTag® Ligand dye. In other studies a HaloTag ® Pull-Down System to study t-SNARE binding activities on the syt1 and Doc2β constructs. (5095)

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Science 361. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. 2018

Chong, S., Dugast-Darzacq, C., Liu, Z., Dong, P., Dailey, G.M., Cattoglio, C., Heckert, A., Banala, S., Lavis, L., Darzacq, X., Tjian, R.

Notes: The authors used Janelia Fluor® 549 HaloTag® and Janelia Fluor® 646 HaloTag® Ligands to stain oncogene fusion knock-ins, allowing visualization of oncogene fusion hubs in the cell nucleus and real-time tracking of individual fusion molecules. (5071)

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4, 11. PLEKHN1 promotes apoptosis by enhancing Bax-Bak hetero-oligomerization through interaction with Bid in human colon cancer. 2018

Kuriyama, S., Tsuji, T., Sakuma, T., Yamamoto, T., Tanaka, M.

Notes: Pleckstrin-homology N1 (PLEKHN1) is involved in reactive oxygen species (ROS) -induced apoptosis, and knockdown of PLEKHN1 in human colon cancer cells lead to increased cell survival. Interestingly, PLEKHN1 expression was absent in cancer patient samples, possibly explaining decreased apoptosis in cancer cells. Magne® HaloTag® Beads were used to pull down PLEKHN1-interacting factors, and an interaction with Bid, a pro-apoptotic protein, was found. PLEKHN1 knockout cells showed loss of Bid localization at the mitochondrial outer membrane and loss of Bak-Bax oligomerization. (5101)

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Nature Methods 15(6), 425–28. Single-shot super-resolution total internal reflection fluorescence microscopy. 2018

Guo, M., Chandris, P., Giannini, J.P., Trexler, A.J., Fischer, R., Chen, J., Vishwasrao, H.D., Rey-Suarez, I., Wu, Y., Wu, X., Waterman, C.M., Patterson, G.H., Upadhyaya, A., Taraska, J.W., Shroff, H.

Notes: The authors combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, and applied it to live samples to achieve rapid, high-contrast super-resolution imaging. Human osteosarcoma U2OS cells were transfected to monitor wild-type Ras dynamics and  imaged with a HaloTag® chimera of HRas labeled with Janelia Fluor 549. (5070)

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Science 356, 1–9. Deconstructing Behavioral Neuropharmacology with Cellular Specificity 2017

Shields, B.C., Kahuno, E., Kim, C., Apostolides, P.F., Brown, J., Lindo, S., Mensh, B.D., Dudman, J.T., Lavis, L.D., Tadross, M.R.

Notes: The authors wanted to manipulate native proteins with cellular specificity in their Parkinson's disease studies. They could spatially restrict drugs to the surface of defined cells using HaloTag® and associated HaloTag® ligand (manufactured for this research in their laboratory), noting that this interaction is covalent, highly efficient and the components are inert. The premise was that the HaloTag® ligand could be fused to a drug by a long flexible linker. If the drug portion had 50% inhibition, then low doses of the drug would result in ligand capture and therefore a high but spatially restricted drug concentration near the HaloTag® protein. (4808)

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J. Immunol. Methods 450, 17-26. Development of NanoLuc® bridging immunoassay for detection of anti-drug antibodies 2017

Nath, N., Flemming, R., Godat, B., and Urh, M.

Notes: This article describes the development of a new bridging immunoassay where NanoLuc® luciferase enzyme is used as an antibody label. (4901)

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Cancer Sci. 109(2), 373–83. PRDM14 directly interacts with heat shock proteins HSP90α and glucose-regulated protein 78. 2017

Moriya, C., Taniguchi, H., Nagatoishi, S., Igarashi, H., Tsumoto, K. and Imai, K.

Notes: PRDM14 is dysregulated in a variety of cancers, including breast and pancreatic cancer, and overexpression leads to stem-cell-like phenotypes associated with aggressive tumors. Here, PRDM14 interacting proteins are identified using the HaloTag® Mammalian Pull-down System. The interactions of PRDM14 and glucoseregulated protein 78 (GRP78) and heat shock protein 90-a (HSP90a) were validated in vivo with the NanoBRET™ assay. (5053)

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Methods 123, 76–88. Quantifying transcription factor binding dynamics at the single-molecule level in live cells. 2017

Presman, D.M., Ball, D.A., Paakinaho, V., Grimm, J.B., Lavis, L.D., Karpova, T.S., Hager, G.L.

Notes: The authors used single-molecule tracking (SMT) approach to follow individual protein molecules in single live cells and performed SMT experiments to learn about the binding characteristics of transcription factors (TFs) such as residence time and bound fractions. They compared JF 549 behavior on HaloTag and other tagging systems, SNAP-tag and CLIP-tag, by labeling all with JF549 dye and studying by whether the fluorophore label and/or tagging system had an effect on data obtained when using the glucocorticoid receptor (GF) as a model TF. Results showed that HaloTag is more resistant to photobleaching and enables tracking of longer binding events compared to SNAP-tag and CLIP-tag. (5069)

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Cell 169, 561. The Right Tool for the Job 2017

Szewczak, L.

Notes: The authors sought to transport small-molecule inhibitors to the surface of neurons to determine whether the small-molecule inhibitors affected cell signaling, and eventually, animal behavior. They fused specific inhibitors to a HaloTag® ligand, thus enriching these inhibitors on the cell surface of interest. Nearby cells lacking the expressed HaloTag® experienced dramatically lowered concentration of inhibitor, and showed no response to it. (4807)

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Nat. Commun. 8, 14259. The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein. 2017

Miyakawa, K., Nishi, M., Matsunaga, S., Okayama, A., Anraku, M., Kudoh, A., Hirano, H., Kimura, H., Morikawa, Y., Yamamoto, N., Ono, A. and Ryo, A.

Notes: Adenomatous polyposis coli protein (APC) is shown to directly interact with HIV-1 Gag protein to stimulate Gag multimerization and spread of viral particles. Direct measurements of the Gag-Gag protein interaction were measured using the NanoBRET™ system. HeLa cells were co-transfected with Gag-HaloTag® and Gag-NanoLuc® expression vectors and BRET signal was monitored after 24 hours. ADC knockdown displayed a substantial decrease in viral production and Gag-Gag interaction. Together, the authors show ADC regulates Gag localization to the PM and viral packaging. (5058)

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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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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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ACS Chemical Biology 11, 2608–17. Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag 2016

Friedman Ohana, R., Levin, S., Wood, M.G., Zimmerman, K., Dart, M.L., Schwinn, M.K., Kirkland, T.A., Hurst, R., Uyeda, H.T., Encell, L.P. and Wood, K.V.

Notes: In this publication, HaloTag® technology was used to identify the protein targets of specific compounds. The tested compounds, kinase inhibitors ibrutinib and BIRB796, were modified with a chloroalkane tag, introduced into cells and after incubation, the cells were lysed. The cellular proteins that interacted with the compounds were captured onto resin with immobilized HaloTag® protein and palladium added to chemically cleave the bound protein from the tagged compound. The protein targets were then identified using mass spectrometry. (4941)

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Cell Rep. 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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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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Nature Methods 12, 244–50. A general method to improve fluorophores for live-cell and single-molecule microscopy. 2015

Grimm, J.B., English, B.P., Chen, J., Slaughter, J.P., Zhang., Z., Revyakin, A., Patel, R., Macklin, J.J., Normanno, D., Singer, R.H., Lionnet, T., Lavis, L.D.

Notes: Using both Janelia Fluor® 549 and 646 HaloTag® Ligands the authors compared them to existing HaloTag® ligands, known to label live cells. The new Janelia Fluor® ligands had superior brightness. (5063)

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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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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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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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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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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 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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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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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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