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. 2020 Jun 16;11(3):e00589-20. doi: 10.1128/mBio.00589-20

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Copyright © 2020 Nkosi et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 5 — A VPS4A dominant negative construct does not impair LMP1 vesicle packaging. HEK293 cells expressing inducible GFP and GFP-tagged wild-type VPS4A (VPS4A WT) or VPS4A-E228Q, which blocks ATP hydrolysis and acts as a dominant negative construct, were transfected with SNAP-LMP1. (A and B) Immunoblot analysis of cell and vesicle lysates for cells expressing GFP, VPS4A, and the mutant VPS4A-E228Q. (C) Semiquantitative Western blot analysis of results from more than 3 independent experiments. Data are presented relative to the value for wild-type LMP1 packaged into EVs. (D to F) HEK293 cells stably expressing shRNAs against ARF6 and c-SRC were constructed and transfected with SNAP-LMP1. (D and E) Whole-cell (D) or EV (E) lysates were separated by SDS-PAGE and analyzed by immunoblot analysis for LMP1 and common EV markers. (F) Semiquantitative Western blot analysis of results from three independent experiments showing relative LMP1 EV secretion (**, adjusted P < 0.0001; ***, adjusted P < 0.05).