Figure 6
From: Dynamic recruitment of ubiquitin to mutant huntingtin inclusion bodies
mHtt IBs sequester active enzymes from the (de)-ubiquitination cascade. (a) Confocal images of Htt-exon1-97Q-C4 protein expressed in transient transfected Neuro-2A cells. 24 hours after transfection cells were electroporated with the Cy5-labeled activity-based probes Ub-Dha, which labels active E1-2-3 enzymes, and Cy5-Ub-PA, which labels active DUBs. One hour after electroporation cells were FlAsH-stained and fixed for imaging. (b) Confocal images of transient transfected Neuro-2A cells co-expressing Htt-exon1-97Q-C4 with GFP-NEDD4.1 and GFP-USP5, respectively. 24 hours after transfection cells were ReAsH-stained and fixed for imaging. Nuclei were stained with DAPI. Scale bar: 6 µm. (c) Filter trap assay (doublets) of mHtt aggregates from Neuro-2A cells transient transfected with the Htt-exon1-97Q-H4 construct for 48 hours. mHtt aggregates sequester endogenous NEDD4 and USP5. Aggregates are stained by anti-HA, anti-Ub, anti-NEDD4 and anti-USP5 antibodies.
