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. 2007 Jun 27;81(18):9812–9824. doi: 10.1128/JVI.01012-07

FIG. 6.

FIG. 6.

KPNA2 interactions with ORF6 and KPNB1. (A) 293 cells were transfected with either Flag-tagged KPNA1, KPNA2, or KPNA2ΔN in combination with HA-tagged ORF6. At 24 h after transfection, cells were treated with 100 IU/ml IFN-β for 30 min, and lysates were collected. Proteins were immunoprecipitated (IP) with anti-Flag antibodies as described in Materials and Methods. Immunoprecipitated extracts were then separated on a 4 to 12% SDS-PAGE gel and analyzed by Western blot (WB) probed with anti-HA, anti-KPNB1, anti-STAT1, and anti-Flag antibodies. Whole-cell extracts (WCL) were also blotted for ORF6 transfection with anti-HA antibodies. (B and C) Vero cells were transfected with STAT1/GFP and either Flag-tagged KPNA2 or KPNA2ΔN. At 24 h posttransfection, cells were treated with 100 IU/ml IFN-β for 60 min before fixation. Proteins were visualized with anti-Flag (αFLAG) antibodies and for the presence of GFP using confocal microscopy. (D and E) Vero cells were transfected as described above (B) except for the addition of HA-tagged ORF6. Cells were stained with anti-Flag and anti-HA antibodies and visualized using confocal microscopy for Flag (Alexa Fluor 546)-, HA (Alexa Fluor 633)-, and GFP-tagged proteins.