CADs do not disrupt the interaction of 19 kDa GP and NPC1.

<p>(A) NPC1-FLAG-enriched LE/Lys membranes from CHO NPC1 cells were disrupted and then incubated with inhibitors for 30 min at RT: mock (4% DMSO), E64d (10 µM), compound 3.47 (13 µM), clomiphene (242 µM), Ro 48-8071 (174 µM), and U18666A (800 µM); each inhibitor was used at a concentration 100 fold over its IC₅₀ for inhibition of infection. The samples were then incubated with 3 µg uncleaved (GP) or cleaved (GP_{19 kDa}) EBOV GP ectodomains for 1 hr at RT. Samples were then lysed, and incubated overnight with anti-FLAG beads. Bound NPC1 and GP were then eluted from beads, and run on an SDS-PAGE gel. The gel was then transferred, blotted for both NPC1 and EBOV GP, and imaged for fluorescent signal. As predicted, uncleaved GP (∼130 kDa) did not co-precipitate with NPC1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone.0056265-Cote1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone.0056265-Miller1" target="_blank">[24]</a>. (B) The intensities of the GP, GP_{19 kDa}, and NPC1 bands from each sample of the blot shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone-0056265-g007" target="_blank">Fig. 7A</a> were quantified and GP or GP_{19 kDa} was normalized to its respective NPC1 band signal. The experiment was conducted four times with similar results, and a representative experiment is shown.