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. 2015 Jan 8;11(1):e1004905. doi: 10.1371/journal.pgen.1004905

Figure 3. APE1L and ROS1 interact in vitro and in tobacco leaves and form a ternary complex with DNA.

Figure 3

(A) Pull-down assay. His-ROS1 was incubated with either MBP or MBP-APE1L bound to an amylose column. After washes, the proteins associated to the resin were separated by SDS-PAGE, transferred to a membrane, and immunoblotted with an antibody against the His-tag. (B) Electrophoretic mobility shift assay. Purified His-ROS1 (75 nM) and increasing concentrations of MBP-APE1L (0, 250, 500, 750 and 1000 nM) were incubated for 15 min at 25°C with a labeled DNA duplex (10 nM) containing a single-nucleotide gap flanked by 3′-phosphate and 5′-phosphate termini. After non-denaturing gel electrophoresis, protein-DNA complexes were identified by their retarded mobility compared with that of free DNA, as indicated. (C) His-ROS1 (75 nM) and MBP-APE1L (1000 nM) were pre-incubated for 4 hours at 15°C with either anti-His or anti-MBP antibodies, and then incubated for 15 min at 25°C with the labeled DNA duplex (10 nM). A control preincubation with anti-PCNA was also performed. Protein-DNA complexes were detected as indicated above. (D) MBP-APE1L (1000 nM) and either His-ROS1 or MBP-ROS1 (75 nM) were incubated during 15 min at 25°C with the labeled DNA duplex (10 nM). Protein-DNA complexes were detected as indicated above. (E) Interaction of APE1L with ROS1 by firefly luciferase complementation imaging assay in Nicotiana benthamiana leaves. Three independent experiments were done with similar results.