Figure 2.

Interaction of the auto‐inhibitory domain of human endonuclease III (hNTH1) with the Y‐box‐binding protein‐1 (YB‐1) protein. (a) Immunoblot against YB‐1 proteins bound to different GST‐hNTH1 affinity Sepahrose beads. Human MCF7 cell extracts (WCE) were incubated with either 50 µg of GST‐hNTH1 constructs or GST‐linked glutathione‐sepharose beads overnight. Proteins bound to the affinity beads were analyzed by SDS‐PAGE with antibodies against YB‐1. (b) IVTT/GST interaction assay. In vitro‐transcribed/translated ³⁵S‐labeled hNTH1 protein fragments bound to YB‐1‐GST affinity beads were separated by SDS‐PAGE and visualized using a PhosphoImager. (c) Schematic representation of the different hNTH1 polypeptides that were used in the hNTH1 affinity chromatography experiments. The black box is the auto‐inhibitory domain and the gray box is the catalytic domain. The amino acid residues of the hNTH1 fragments used in this study are indicated on the left. YB‐1 binding is indicated on the right by the ‘+’ sign. The ‘–’ sign indicates no binding detected.