Effect of mutation of the GDNQ motif on expression and intracellular localization of EBOV L.
(A) Western blot of 293T cell lysates transfected with L-mCherry or Lmut-mCherry. Blots were cut in half, and stained with monoclonal antibodies against mCherry or actin. (B) Quantification of L-mCherry expression. Results from western blotting were quantified, and the expression level of wild-type L set to 100%. Means and standard deviations of 4 biological replicates from 3 independent experiments are shown. (C) Intracellular localization of L-mCherry or Lmut-mCherry. Cells were transfected with expression plasmids for all RNP proteins (including either wild-type L, L with a fluorescent mCherry tag (L-mCherry) or a mutated version of L-mCherry without the GDNQ motif (Lmut-mCherry)), a GFP-expressing replication-competent minigenome, T7-polymerase to facilitate initial transcription of minigenome vRNA, and pmTurquoise2-H2A to label cell nuclei. Live cells were visualized after 48 hours by spinning disc confocal microscopy. The white bar indicates 10 μm.