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. 2014 Apr 29;289(23):16223–16238. doi: 10.1074/jbc.M113.527424

FIGURE 3.

FIGURE 3.

UHRF1 is PARylated preferentially onto its SRA domain but also onto its TTD. A, PARylation of full-length UHRF1 by PARP1. Myc-UHRF1 (lanes 1, 2, 5, and 6) or Myc-TRF2 (lanes 3, 4, 7, and 8) was expressed in COS-1 cells, immunopurified with an anti-Myc antibody, and incubated together with either PARP1 or without PARPs in activity buffer containing [α-32P]NAD+ and fragmented DNA. Right panel, autoradiography. Left panel, analysis of the fusion proteins with an anti-Myc antibody by Western blotting. B, upper panel, schematic representation of UHRF1 indicating the PARylated TTD and SRA domain. Lower panel, GFP fusion proteins expressing GFP alone (lanes 1 and 7) or different domains of UHRF1 (GFP-Ring (lanes 2 and 8), GFP-SRA (lanes 3 and 9), GFP-PHD (lanes 4 and 10), GFP-Ubl (lanes 5 and 11), and GFP-TTD (lanes 6 and 12)) were expressed in COS-1 cells, immunopurified by GFP trapping, and incubated together with PARP1 in the activity buffer containing [α-32]PNAD+ and fragmented DNA as in A. Left, analysis of the PARylated domains by autoradiography. The upper band represents PARylated PARP1. Right, analysis of the fusion proteins with an anti-GFP antibody by Western blotting.