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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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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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Oncol. Lett. 9, 330-4. Alternative splicing isoform in succinate dehydrogenase complex, subunit C causes downregulation of succinate-coenzyme Q oxidoreductase activity in mitochondria. 2015

Satoh, N., Yokoyama, C., Itamura, N., Miyajima-Nakano, Y., and Hisatomi, H.

Notes: Total RNA was isolated from HTC-15 human colorectal carcinoma cells with the ReliaPrep™ RNA Cell Miniprep System. Isolated RNA was used in an RT-PCR method to amplify alternative splice variants of the target transcript. Once identified, a RT-qPCR experiment was designed to look for the major splice variants in normal cell lines derived from 15 human tissues (RNA isolation details not provided, assumed to be the same as HTC-15). The alternative splice variants were overexpressed in HTC-15 cells by transfection via FuGENE® HD Transfection Reagent and noted a decline in full-length expression. (4607)

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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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Comb. Chem. High Throughput Screen 17, 12–24. A high content assay to assess cellular fitness. 2014

Antczak, C., Mahida, J.P., Singh, C., Calder, P.A. and Djaballah, H.

Notes: Researchers were interested in developing a protocol to test transfection efficiency, cellular toxicity and stress response of transfection reagents. Using an EGFP DNA plasmid transfected into HeLa cells as their model system, seven different transfection reagents including FuGENE® HD Transfection Reagent were assessed using whole well imaging, Hoechst staining of nuclei and immunostaining with multiplexed readout. The transfection reagents were tested in parallel at 0.025, 0.05 and 0.1μl/well using EGFP plasmid or scrambled siRNA duplex, and compared to negative control wells (no nucleic acid). Transfection efficiency and cellular effects including morphology were assessed 24, 48 and 72 hours post transfection. Of the panel of reagents, FuGENE® HD Transfection Reagent had the highest transfection efficiency and according the authors “emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening”. (4766)

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J. Bacteriol. 195, 708–16. Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments. 2013

Jiwani, S. et al.

Notes: The authors transiently transfected 2 × 105 HeLa cells grown on coverslips in a 6-well plate using the FuGene® HD Transfection Reagent, 2.5µg of DNA and a reagent:DNA ratio of 8:2.5. (4433)

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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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mBio. epub, e00418–12. Sensing of bacterial type IV secretion via the unfolded protein response 2013

de Jong, M.F., Starr, T., Winter, M.G., den Hartigh, A.B., Child, R., Knodler, L.A., van Dill, J.M., Celli, J. and Tsolis, R.M.

Notes: FuGENE® HD Transfection Reagent was used to transiently transfect HeLa cells seeded on 12mm coverslips in 24-well plates. Cells were seeded at a concentration of 5 × 104 cells/well and transfected after 24 hours using a 2:1 ratio of FuGENE® HD reagent to DNA (4µl of reagent, 2µg of DNA). Transiently transfected cells were used for confocal microscopy. For luciferase assays, HEK293 cells were seeded into 48-well plates at 40% confluency, and 24 hours later, FuGENE® HD reagent was used to transfect the cells with one of a variety of constructs. (4411)

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J. Cell Biol. 202, 579–95. A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. 2013

Britton, S. Coates, J. and Jackson, S.P.

Notes: The authors transiently transfected 5 × 106 HEK293-AAV cells per 100cm dish using the FuGene® HD Transfection Reagent, 9µg of DNA and a reagent:DNA ratio of 3:1. (4431)

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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. Virol. 87, 2109–19. N-terminal phosphorylation sites of herpes simplex virus 1 ICP0 differentially regulate its activities and enhance viral replication. 2013

Mostafa, H.H., Thompson, T.W. and Davido, D.J.

Notes: In this paper, the FuGENE® HD Transfection Reagent was used to transiently transfect Vero, Hep-2 and HeLa cells. Transfection conditions were as follows: Vero cells plated in 60mm dishes at 4 × 105 cells per plate and transfected with 3.5µg of DNA; Vero cells plated on 24-well plates at 5 × 104 cells/well and transfected with 1µg DNA; Hep-2 cells plated in 12-well plates at 5 × 104 cells/well on glass cover slips and transfected with 1µg of DNA; Hep-2 cells plated in 24-well plates at 5 × 104 cells/well on glass cover slips and transfected with 0.5µg and 1µg of DNA; HeLa cells plated in 24-well plates at 5 × 104 cells/well and transfected with 1µg of DNA. A 3:1 ratio of reagent to DNA was used for all transfections. (4419)

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Genome Res. 23, 800-11. Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay. 2013

Kheradpour P, Ernst J, Melnikov A, Rogov P, Wang L, Zhang X, Alston J, Mikkelsen TS, Kellis M.

Notes: HepG2 cells were plated, 5 × 106 cells in 15cm plates. Transfections were performed 24 h later using Fugene HD. The authors used 15 μg of DNA per transfection and a Fugene:DNA ratio of 7:2. (4425)

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Genome Res. 23, 800–11. Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay. 2013

Kheradpour, P. et al.

Notes: The authors transiently transfected 5 × 106 HEK293 cells per 150cm dish using the FuGENE® HD Transfection Reagent, 15µg of DNA and a reagent:DNA ratio of 7:2. (4432)

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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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J. Virol. 86, 8198–8209. Cellular GCN5 is a Novel Regulator of Human Adenovirus E1A-Conserved Region 3 Transactivation 2012

Ablack, J.N.G., Cohen,M., Thillainadesan, G., Fonseca, G.J., Pelka, P., Torchia, J. and Mymryk, J.S.

Notes: FuGENE HD reagent was used for transient transfection of A549 cells and wild type mouse embryonic fibroblasts. A 3:1 ratio of reagent to DNA was used in the transfection reactions (9µl reagent, 3µg DNA). (4410)

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Sci. Signal. 5, ra64. FAM123A binds to microtubules and inhibits the guanine nucleotide exchange factor ARHGEF2 to decrease actomyosin contractility 2012

Siesser, P.F., Motolese, M., Walker, M.P., Goldfarb, D., Gewain, K., Yan, F., Kulikauskas, R.M., Chien, A.J., Wordeman, L. and Major, M.B.

Notes: To better understand what roles FAM123A may play in signaling, cell behavior and human disease, HT1080 sarcoma cells were plated on MatTek dishes coated with 5mg/ml fibronectin before transfection with Venus (a yellow fluorescent protein)-tagged FAM123A or Venus-WTX, another member of the FAM123 gene family, using FuGENE® HD Transfection Reagent. The cells were imaged the next day for low-resolution analysis. For a higher magnification, dynamic analysis, HT1080 cells were plated onto Delta T dishes coated with 5mg/ml fibronectin and transfected with EGFP-FAM123A and FuGENE® HD Transfection Reagent. The next day, cell images were captured every 10 seconds. Examining the effect of silencing FAM123A, a reporter assay used the pGL4.34[luc2P/SRFRE/Hygro] Vector cotransfected with a CMV-Renilla coreporter and other effector plasmids or siRNA, and luciferase levels assessed using the Dual-Glo® Luciferase Assay System. (4246)

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FASEB J. 26, 1629-39. Influenza A virus inhibits cytoplasmic stress granule formation. 2012

Khaperskyy DA, Hatchette TF, McCormick C.

Notes: Fugene HD was used with HeLa-TetOff cells, seeded at a density of 100,000 cells/well of a 12-well culture plate. On day 2, cells were transfected with 0.5 μg total DNA/well and FuGene HD reagent with a DNA to FuGENE HD ratio of 1:3 (w/v). (4423)

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Mol. Biol. Cell 23, 3499-510. Large G3BP-induced granules trigger eIF2α phosphorylation. 2012

Reineke, L.C., Dougherty, J.D., Pierre, P., and Lloyd, R.E.

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect HeLa and MEF cells. Transfection conditions were as follows: HeLa Tet-On cells were plated on glass coverslips at 1-1.3 X 10e5 cells/well in 12-well plates 1 day prior to transfection with 0.2µg plasmid DNA. Mouse embryonic fibroblast (MEF) cells were plated at 5x10e4 cells/well in 12-well plates 1 day prior to transfection with 2µg plasmid DNA.



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Development 139, 3986-96. Pancortins interact with amyloid precursor protein and modulate cortical cell migration. 2012

Rice, H.C., Townsend, M., Bai, J., Suth, S., Cavanaugh, W., Selkoe, D.J., and Young-Pearse, T.L.

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect HEK293 cells. HEK293 cells were plated at 1 X 10e6 cells/well in 6-well plates prior to transfection. (4413)

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J. Virol. 86, 1193-1202. Varicella-zoster virus inhibition of the NF-κB pathway during infection of human dendritic cells: role for open reading frame 61 as a modulator of NF-κB activity. 2012

Sloan, E., Henriquez, R., Kinchington, P.R., Slobedman, B., and Abendroth, A.

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect 293FT human embryonic kidney cells. Transfection conditions were as follows: Cells were grown to 30% confluency in 6-well plates prior to transfection with 3µg DNA at a 3:1 FuGENE® reagent:DNA ratio. (4415)

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J. Virol. 86, 1193–202. Varicella-zoster virus inhibition of the NF-κB pathway during infection of human dendritic cells: role for open reading frame 61 as a modulator of NF-κB activity. 2012

Sloan, E., Henriquez, R., Kinchington, P.R., Slobedman, B. and Abendroth, A.

Notes: The authors examined the effect of Varicella-zoster virus (VZV) on the NF-κB pathway and found inhibition of the pathway in VZV-infected dendritic cells. To determine which open reading frame (ORF) in the virus was responsible, seven hemagglutatin-tagged ORFs were cloned and transfected into 30% confluent 293FT human embryonic kidney cells (seeded 24 hours before) using FuGENE® HD Transfection Reagent with a 2:6 DNA:reagent ratio. After 48 hours, the cells were harvested for use in flow cytometry or Western blot analysis. An NF-κB reporter assay used 293FT cells seeded in six-well plates at 30% confluency and transfected 2µg of NF-kB GFP reporter vector and 1µg of VAV ORF expression constructs with FuGENE® HD Transfection Reagent. After 24 hours, 20nM of TNF-α was added and the cells incubated another 24 hours before harvest and analysis by flow cytometry. (4245)

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Mol. Biol. Cell 23, 864–80. Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content. 2012

Mundy, D.I., Li, W.P., Luby-Phelps, K. and Anderson, R.G.

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect SV589 Human Fibroblast cells. Transfection conditions were as follows: Cells were grown in 35mm plates and transfected with 2µg DNA at a 3:1 FuGENE® reagent:DNA ratio. (4422)

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J. Biol. Chem. 287, 36370–83. Human Pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs. 2012

van Etten, J. et al.

Notes: The authors transiently transfected 2 × 104 HEK293 cells per well of a 96-well plate using the FuGene® HD Transfection Reagent, 0.1µg of DNA and a reagent:DNA ratio of 3:1. (4430)

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PLoS Genet. 8, e1002941. MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells. 2012

Goodier, J.L., Cheung, L.E. and Kazazian, H.H. Jr.

Notes: To better understand the role MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), in reducing retrotransposon activity, 293T human embryonic kidney cells expressing MOV10 and one of three retrotransposons (LINE1 (L1), Alu or SVA) were lysed in a buffer that included RNasin® Ribonuclease Inhibitor, and FLAG-tagged L1 complexes immunoprecipitated and analyzed by mass spectrometry. Several retrotransposon assays were conducted using FuGENE® HD Transfection Reagent for transfected constructs into 293T, HeLa and HeLa-HA cells. (4255)

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