Fig. 4.

VCP adaptors undergo dynamic exchange during IP of untagged endogenous VCP. A, VCP antibody does not bind ND1L. Full-length VCP and the ND1L fragment were fractionated by SDS-PAGE and subjected to Western blotting with anti-VCP. Left panel: Ponceau S staining of the nitrocellulose filter. Right panel: Western blot of the same filter. B, Schematic workflow of “Sponge” experiment. C, Effect of ND1L competitor on recovery of VCP-interacting proteins. The indicated amounts of purified ND1L were added to 1 milligram of HEK293 cell lysate, which was then subjected to IP for 1 min with anti-VCP followed by 5 min with protein A/G resin prior to mass spectrometry. n = 1 for all treatments. D, The relative amounts of individual VCP adaptors identified in the experiment in panel B are plotted. Relative protein amounts were estimated by LFQ value from MaxQuant.