Fig. 2: In cellula activity-based E3 ligase profiling.
From: ATP functions as a pathogen-associated molecular pattern to activate the E3 ubiquitin ligase RNF213
a In cellula activity-based E3 ligase profiling involving the delivery of a biotin-tagged E2~Ub ABP into live cells by electroporation. Labeled E3s can be subsequently enriched and analysed by mass spectrometry or Western blot. The abundance of a recovered E3 is a proxy for its transthiolation activity, allowing relative changes to be inferred. b Western blot demonstrating intracellular delivery of ABP by electroporation followed by subsequent incubation for the specified time. Following lysis, the ABP and its labeled proteins can be subsequently captured with streptavidin resin. Panel shown representative of n = 3. c Mass spectrometry analysis showing total spectral counts from E3s and the two E1 enzymes (UBA1 and UBA6) enriched by Biotin-ABP electroporated into 293T cells. Plotted proteins had >10 times more spectral counts 1 h post-electroporation than ABP (based on UBE2D3) treatment without electroporation. E3s were selected based on Pfam domain terms. The E3 coverage was similar whether ABP was incubated for 1 or 4 h post-electroporation. Source data are provided as a Source Data file.
