FIGURE 5.

Screen of 41 human DUBs indicates USP9X to be the counteracting DUB of UBE3A. (A) 41 different human DUBs were silenced using 10 nM siRNAi. After GFP-pulldown of DDI1-GFP, FLAG/GFP intensity ratios were determined. Those ratios were normalized to the average of three lowest values on each 6-well experimental dataset. Upon silencing of most DUBs, the ubiquitination of DDI1 did not change quantitatively (0.5 < fold-change < 2, white). However, silencing of some DUBs decreased DDI1 ubiquitination (fold-change < 0.5, red), while some others increased it (fold-change > 2, gray). Black bars highlight the DUBs that affect more severely DDI1 ubiquitination (UCHL-5, USP7, USP9X, and USP42). (B) siRNAs were used to silence UCHL-5, USP7, USP9X, and USP42 DUBs and scramble siRNAi was used as a control. After GFP-pulldown, DDI1 ubiquitination was detected by western blot analysis using anti-FLAG (red) and anti-GFP (green) antibodies. Equivalent amounts of ectopically overexpressed ubiquitin was detected in the cell lysates [Input FLAG-(Ub)], while USP9X silencing was corroborated by anti-USP9X antibody (USP9X). Quantification was performed with Image-J after normalizing FLAG/GFP intensities. USP9X inhibition significantly enhanced DDI1 ubiquitination [one-way ANOVA, ^∗∗p-value < 0.05, (mean ± S.E.M., n = 3)]. DDI1 ubiquitination also increased upon USP42 silencing, but not significantly, while no changes were observed for the UCHL-5 and USP7 experiments.