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. Author manuscript; available in PMC: 2017 Nov 20.
Published in final edited form as: J Mol Biol. 2016 Sep 24;428(23):4639–4650. doi: 10.1016/j.jmb.2016.09.018

Figure 1. RNF4-catalyzed ubiquitination of a polySUMO substrate by UBCH5B and RAD6B enzymes.

Figure 1

(a) Time course comparing the in vitro ubiquitination of 4xSUMO2, using both native and methylated Ub, by RNF4 in the presence of UBCH5B. Reactions contained 200 nM E1, 5 μM E2, 1 μM RNF4, 15 μM 4xSUMO-2 and 100 μM of native or methylated Ub, and were incubated at 37°C. Samples were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue stain. (b) Time course comparing the in vitro ubiquitination of 4xSUMO2, using both native and methylated Ub, by RNF4 in the presence of RAD6B. Reactions contained 200 nM E1, 5 μM E2, 1 μM RNF4, 15 μM 4xSUMO-2 and 100 μM of native or methylated Ub, and were incubated at 37°C. Samples were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue stain.