Supplementary Figure 1: Stimulation of RNF4-mediated synthesis of unanchored K63-linked ubiquitin chains by SUMO-2 chains and biochemical analysis of the isopeptide linked Ubc13~Ub conjugate.
From: Structural basis for the RING-catalyzed synthesis of K63-linked ubiquitin chains
(a) Coomassie-blue stained SDS-PAGE (top panel) and Western blot (bottom panel) analysis of ubiquitination assays with and without 4xSUMO-2. A negative control without ATP is shown with 4xSUMO-2 present. Time points are denoted above the gels.
Ubc13 C87K was used to form a stable isopeptide linked Ubc13~Ub conjugate for crystallography. Due to the identification of non-specific ubiquitination of Ubc13 K92 in the Ubc13 C87A negative control, K92A was also introduced in combination with C87K to prevent non-specific ubiquitination of Ubc13.
(b) Coomassie-blue stained SDS-PAGE analysis of the ability of Ubc13 C87K and Ubc13 C87K K92A to form an isopeptide linked conjugate with ubiquitin. Negative controls containing the C87A mutation were tested in parallel. Time points are shown above the gels.
(c) Coomassie-blue stained SDS-PAGE analysis of ubiquitination assays showing the effect of the introduction of K92A on Ubc13 activity compared to WT Ubc13. Reactions contained 0.1 µM E1, 1 µM Ubc13, 1 µM Ube2V2, 0.55 µM RNF4, 5.5 µM Ub~4xSUMO-2, 20 µM ubiquitin, 3 mM ATP, 5 mM MgCl2, 50 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM TCEP, and 0.1 % NP40. Reactions were incubated at 37 °C. Time points are shown above the gels. The red asterisk indicates formation of di-ubiquitin.
(d) The chromatogram shows separation of the Ube2V2–Ubc13~Ub complex and free ubiquitin by size exclusion chromatography. Elution fractions analysed by SDS-PAGE are denoted with magenta, green and black bars below the chromatogram.
(e) Coomassie stained SDS-PAGE analysis of size exclusion chromatography showing input to the chromatography numbered 1 – 3 and analysis of the elution fractions highlighted in d.
.
