FIG 3.

Knockdown of upstream ESCRT-dependent proteins reduces LMP1 vesicle secretion and packaging. HEK293 cells expressing inducible scramble, Alix, Hrs, Syntenin-1, or TSG101 shRNA constructs were transfected with GFP-LMP1. (A and B) Whole-cell (equal mass loaded) (A) and EV (equal volume loaded) (B) lysates were separated by SDS-PAGE and analyzed by immunoblot analysis for LMP1 and common EV markers (Alix, Hrs, calnexin, HSC70, TSG101, Syntenin-1, Flotillin-2, and CD63). (C) Semiquantitative Western blot analysis of results from more than 3 independent experiments. The data are presented relative to the values for wild-type LMP1 packaged into EVs. (D and E) EVs harvested from HEK293 cells expressing shRNAs transfected with GFP-LMP1 were analyzed by nanoparticle tracking analysis for size (D) and quantity (E). (F) HEK293 cells expressing the shRNAs and the NF-κB luciferase reporter were transfected with LMP1 and evaluated for activation of NF-κB signaling (*, adjusted P < 0.005; **, adjusted P < 0.0001; ***, adjusted P < 0.05; ns, not significant).