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Fig. 5 | Epigenetics & Chromatin

Fig. 5

From: The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase

Fig. 5

SHPRH recruits UBE2D1 to, and forms stable complexes with, nucleosomes. a Western blot of SHPRH and UBE2D1 immunoprecipitations. FLAG-tagged versions of SHPRH and UBE2D1 were overexpressed in HEK293 cells. Whole cell extracts were then used to immunoprecipitate the two proteins using specific antibodies, as indicated on top. IgG was used as a negative control. The input (IN), the flow-through (FT) and the immunoprecipitates (IP) were probed using antibodies (α) against SHPRH and UBE2D1, as indicated on the right. b Electrophoretic mobility shift assay. SHPRH (20 nM) was incubated with radiolabeled mononucleosomes (20 nM; lane 2), or, in addition, with UBE2D1 (800 nM; lane 3). As controls, we used mononucleosomes alone (20 nM; lane 1) or incubated in the presence of UBE2D1 or BSA (800 nM; lanes 4 and 6, respectively) or, SHPRH + BSA (lane 5), as indicated on top. Reactions were separated by native PAGE and visualized using a phosphorimager screen. The positions of free (unbound) nucleosomes and protein-nucleosome complexes are indicated by arrows, on the right

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