Fig. 4

SHPRH is a histone/nucleosome E3-ligase. a E2-conjugating enzyme screen. Twenty-six different E2s (as indicated on top of the panels) were tested for their ability to support free or nucleosomal histone ubiquitination by the SHPRH E3-ligase. Histone octamers purified from HeLa cells (upper panel) or nucleosome arrays (lower panel) were used as substrates for ubiquitination. SHPRH (85 nM) and the substrates (600 ng histones or nucleosome arrays per 20 μl reaction) were added to a mix containing E1, E2-conjugating enzymes and ubiquitin, and the reactions were incubated for 1 h at 37 °C prior to adding SDS loading buffer to stop the reactions. Samples were then boiled and the proteins were separated on a 15% PAA gel followed by Western blotting against histone H3. b the same samples as in (a) were blotted using an antibody against histone H4. (Parts of gels from the same experiment were aligned using Photoshop for clarity). Asterisks denote non-specific Western Blot signals (based on the Coomassie-stained E2 gel provided by Boston Biochem)