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. 2013 Jun 13;288(31):22621–22635. doi: 10.1074/jbc.M113.481382

FIGURE 5.

FIGURE 5.

Co-IP analysis of DENV-2 and -4 NS5 interactions. A, HEK293 cells were co-transfected with either a plasmid coding for KPNA2-V5 or no plasmid (con) together with pcDV2-NS5F (DV2-NS5F), pcDV4-NS5F (DV4-NS5F), or pc3xFLAG-Rac1 (Rac1-F). Co-IP analysis was done using anti-V5 beads. The immunoprecipitates (IP) and total cell lysates (Input) were analyzed by SDS-PAGE and Western blotting using an anti-FLAG antibody to detect NS5F and Rac1-F and an anti-V5 antibody to detect KPNA2-V5. *, nonspecific band recognized by the anti-V5 antibody. B, HEK293 cells were co-transfected with either pGFP-DV2-NS5F (GFP-DV2-NS5F), pGFP-DV4-NS5F (GFP-DV4-NS5F), or pEGFP (GFP) together with a plasmid coding for human STAT2. Co-IP analysis was conducted using GFP-Trap beads and TBS containing 150 mm NaCl and 1% Triton X-100 for cell lysis and washing. The immunoprecipitates (IP) and total cell lysates (Input) were analyzed by SDS-PAGE and Western blotting using anti-STAT2 and anti-GFP antibodies to detect STAT2 and GFP-NS5/GFP, respectively. All co-IP assays were done 24 h post-transfection.