FIG 1.

Analysis of the interaction between NS4B and NS3 by coimmunoprecipitation (co-IP) and proximity ligation assay (PLA). (A and B) BHK-21 cells were infected with DENV-2 at an MOI of 1. At 48 h p.i., the infected cells were subjected to co-IP (A) and PLA (B) as described in Materials and Methods. (C) 293T cells were cotransfected with expression vectors pXJ-NS2B-NS3 and/or pXJ-2K-NS4B. At 48 h p.t., the transfected cells were subjected to co-IP. (D) BHK-21 cells were cotransfected with expression vectors pXJ-NS2B-NS3 and/or pXJ-2K-NS4B. At 48 h p.t., the transfected cells were analyzed by PLA. BHK-21 cells, rather than 293T cells, were chosen for the PLA because PLA requires many washing steps, and 293T cells are easily washed off during these steps. In the PLA, red fluorescent signals indicate NS3-NS4B proximity. Nuclei were stained in blue by 4′,6-diamidino-2-phenylindole (DAPI).