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. 2011 Feb 10;7(2):e1001278. doi: 10.1371/journal.ppat.1001278

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Popescu et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Schematic representation of the constructs used in this study. NS2-GFP corresponds to the transmembrane domain of NS2 in fusion with GFP, whereas NS2-GTM corresponds to the transmembrane domain of VSV-G protein in fusion with the cytosolic domain of NS2. All the proteins contain a HA tag at their N-terminus. (B) HA-NS2 co-immunoprecipitates with p7-Flag. 293T cells were transfected with plasmids expressing p7-Flag, HA-NS2 from different genotypes, HA-NS2 mutants or control plasmids. At 24h post-transfection cells were lysed and immunoprecipitations with an anti-Flag antibody were performed. The immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with an anti-HA antibody to identify the presence of co-immunoprecipitated HA-NS2. The presence of HA-NS2 in transfected cells was confirmed by Western blotting. The actin content was also analyzed to verify that equal amounts of cell lysates have been used.