Fig. 3: BI8626 is an efficient and selective substrate of HUWE1HECT.
From: Selective ubiquitination of drug-like small molecules by the ubiquitin ligase HUWE1
a SEC-based analyses of HUWE1HECT-driven ubiquitination reactions, containing the indicated BI8626 concentrations, monitoring the mono- and diubiquitinated BI8626 by absorbance at 340 nm. b Multi-turnover HUWE1HECT-driven ubiquitination reactions ±20 μM purified mono-ubiquitinated BI8626 (BI8626-Ub), followed by a fluorescent Ub tracer. The bottom part of the gel is shown at a different exposure to allow for diUb and diUb-modified BI8626 to be distinguished. For protein input, see Supplementary Fig. 12b. c Quantification of assays as shown in (b). For each experiment, the amount of HUWE1HECT autoubiquitination at 30 minutes was normalized to that under inhibitor-free (‘untreated’) conditions. Data are represented as mean ± SD (n = 3 biological replicates). Statistical analysis using un unpaired, two-sided t-test (ns (not significant): p \(\ge\) 0.05). d SEC-based analyses of BI8626 ubiquitination, driven by the indicated HECT domains, monitoring the mono- and diubiquitinated BI8626 by absorbance at 340 nm. e Quantification of BI8626 monoubiquitination by the indicated HECT domains from SEC analyses as shown in (d). The signal produced by HUWE1HECT was normalized to 1. Data are represented as mean ± SD (n = 2 biological replicates). Source data are provided as a Source Data file.
