FIG 5.

NS1 enhances MLKL oligomerization and membrane translocation. (A) HEK293T cells were cotransfected with the Flag-tagged MLKL-expressing plasmid and increasing amounts of the NS1-expressing plasmid. At 36 h posttransfection, cell lysates were separated into the total, cytoplasmic (C), or membrane (M) fraction. The cell lysates were resolved on SDS-PAGE gels with (reducing) or without (nonreducing) β-mercaptoethanol and analyzed by Western blotting using the indicated antibodies. (B) HEK293T cells were cotransfected with the Flag-tagged MLKL-expressing plasmid and either the WT NS1- or NS1(R38A/K41A)-expressing plasmid. At 36 h posttransfection, cell lysates were harvested and subjected to assays as described above for panel A. (C) Differentiated THP1 cells were either mock infected or infected with PR8 virus at an MOI of 10. At 8 h p.i., cell lysates were harvested and subjected to assays as described above for panel A.