Application number: P201030204. ligands. Conclusions/Significance Our protein redistribution method does not present the architectural requirement of re-constructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only cell transfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus of living cells to test protein-protein interactions or to perform functional studies by protein ligand sequestration. Introduction Viroplasms, viral factories or virus inclusion bodies are different names given to the cellular compartments where most viruses carry out their morphogenesis. They are usually generated from one or several viral proteins that act as a scaffold or matrix, nucleating the inclusion that is formed by protein-protein interactions. The matrix proteins attract and concentrate the viral components, increasing the overall efficiency of the viral replication process [1], [2]. Avian reoviruses belong to the genus (EcoRI site is single underlined and the start codon and SV40 T antigen NLS is double underlined) and the reverse primer was (XbaI Miglustat hydrochloride site is single underlined and the stop codon is double underlined). The PCR product was digested and cloned into the EcoRI and XbaI sites of pCDNA3.1/Zeo to generate pCDNA3.1/Zeo-TAg-NLS-muNS. ii) TAg-NLS-GFP-muNS or TAg-NLS-GFP-muNS-Mi To express the SV40 T antigen NLS fused to the N terminus of either GFP-muNS or GFP-muNS-Mi, the recombinant plasmids pEGFP-C1-M3 [13] and pEGFP-C1-M3(448C635) [14] were subjected to PCR amplification with the following primers: the forward primer was (BamHI site is single underlined and the start codon and SV40 T antigen NLS is double underlined), and the reverse primer was Miglustat hydrochloride (XbaI site is single underlined and Rabbit Polyclonal to IL4 the stop codon is double underlined). PCR products were digested and cloned into the BamHI and XbaI sites of pCDNA3.1/Zeo to generate either pCDNA3.1/Zeo-TAg-NLS-GFP-muNS or pCDNA3.1/Zeo-TAg-NLS-GFP-muNS-Mi. iii) TAg-NLS-muNS-Mi To express the SV40 T antigen NLS fused to the N terminus of muNS-Mi, the recombinant plasmid pGEMT-M3 [13] was subjected to PCR amplification with the following primers: the forward primer was (BamHI site is single underlined and the start codon and SV40 T antigen NLS is double underlined), and the reverse primer was (XbaI site is single underlined and the stop codon is double underlined). The PCR product was digested and cloned into the BamHI and XbaI sites of pCDNA3.1/Zeo to generate pCDNA3.1/Zeo-TAg-NLS-muNS-Mi. iv) VP16-GFP-muNS or VP16-GFP-muNS-Mi To generate the recombinant plasmids pVP16-GFP-muNS and pVP16-GFP-muNS-Mi, which express a transcriptional activation domain (VP16) fused to the N terminus of either GFP-muNS or GFP-muNS-Mi, the recombinant plasmids pEGFP-C1-M3 [13] and pEGFP-C1-M3(448C635) [14] were subjected to PCR amplification with the Miglustat hydrochloride following primers: the forward primer was (BamHI site is single underlined and the start codon is double underlined), and the reverse primer was (XbaI site is single underlined and the stop codon is double underlined). PCR products were cut with BamHI and XbaI and ligated to pVP16 (Clontech, Saint Germain en Laye, Francia) that had been cut with the same enzymes. v) VP16-muNS-Mi To generate the recombinant plasmid pVP16-muNS-Mi, which expresses a transcriptional activation domain (VP16) fused to the N terminus of muNS-Mi, the recombinant plasmid pGEMT-M3 [13] was subjected to PCR amplification with the following primers: the forward primer was (EcoRI site is single underlined), and the reverse primer was (XbaI site is single underlined and the stop codon is double underlined). The PCR product was cut with EcoRI and XbaI and ligated to pVP16 (Clontech, Saint Germain en Laye, Francia) that had been cut with the same enzymes. (EcoRI site is single underlined), and the reverse primer was: (XbaI site is single underlined and the stop codon is double underlined). The PCR product was digested and cloned into the EcoRI and XbaI sites of pM (Clontech, Saint Germain en Laye, Francia) that had been cut with the same enzymes. The correctness of the constructs was confirmed by sequencing and Western blot analysis of the expressed proteins. Acknowledgments We thank Juan Bautista Zalvide for donating plasmid pCMV-wtTAg, Mark van Raaij for critical reading of the manuscript and Leticia Barcia Castro for excellent technical assistance. We also thank Laboratorios Intervet (Salamanca, Spain) for providing pathogen-free embryonated eggs. Footnotes Competing Interests: The results presented in this manuscript are patent pending. Application number: P201030204. Inventors: Alberto Brandariz-Nu?ez, Rebeca Menaya Vargas, Javier Benavente and Jose Martinez-Costas. Country: Spain. All the materials described will be available for research purposes. This does not alter the.