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. 2017 May 17;8(30):49655–49670. doi: 10.18632/oncotarget.17912

Figure 4. PACT interacts with the N proteins of MHV and SARS-CoV.

Figure 4

(A) HEK-293T cells were transfected with expression plasmids encoding HA-tagged PACT and Flag-tagged MHV or SARS-CoV N proteins. At 28 h after transfection, cells were then fixed for IFA to detect PACT (green) and N proteins (red) with anti-HA and anti-Flag antibodies, respectively. DAPI (blue) indicates the locations of the cell nuclei. Fluorescent images were acquired with a confocal laser scanning microscope (Olympus Fluoview ver. 3.1, Japan). (B and D) HEK-293T cells were transfected with expression constructs encoding HA-tagged PACT and Flag-tagged MHV or SARS-CoV N proteins. The cells were lysed at 28 h after transfection and subjected to immunoprecipitation with anti-HA antibody. The whole-cell lysates (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting using anti-Flag, anti-HA, or anti-β-actin antibodies. (C and E) Transient transfection and immunoblotting analyses were carried out in the manner described in B and D above, but immunoprecipitation was performed with anti-Flag antibody.