Fig. 3: Efficiency of detection of substrate candidates by difference in the fusion site between TUBE and E3.

a Construction of an N-terminal or C-terminal FLAG–TUBE-fused probe. b, c Venn diagram for identified substrate candidates in Parkin experiments with CCCP (10 μM) treatment for 1 h in HeLaS3 cells stably expressing each probe (b) and in TRIM28 experiments in HEK293T cells stably expressing each probe (c). A probe in which TUBE was fused to the N or C terminus of Parkin or TRIM28 was used. d, e Substrate candidates for Parkin (d) and TRIM28 (e). Relative label-free quantification (LFQ) abundance is indicated by the color scale. Proteins with PSMs of >3 in at least one experiment (Exp) and >1 in at least two experiments were considered as substrate candidates for each E3 ligase. f, g Detection of ubiquitinated endogenous substrates. Cells expressing the N-terminal or C-terminal FLAG–TUBE-fused probe were harvested and anti-FLAG immunoprecipitates were analyzed by immunoblotting. Vertical bars and arrows denote the positions of ubiquitinated substrates and unmodified substrates, respectively.