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. 2003 Jun 1;31(11):2717–2724. doi: 10.1093/nar/gkg385

Figure 2.

Figure 2

The NLS of MPG is essential for nuclear translocation. (A) Purified GST–importin α and GST–importin β were coupled onto GST-Sepharose resin and incubated in batch with MPG/DNA or MPGΔNLS/DNA complexes for 1 h, extensively washed and incubated with 50 nM glutathione to elute the complexes. DNA was then extracted and analysed by agarose gel electrophoresis (0.8%). Lane 1, control DNA; lane 2, free plasmid does not associate with importin α; lane 3, binding of MPG/DNA to importin α; lane 4, binding of MPG/DNA to importin β; lane 5, MPG/DNA does not associate with importin β. (B) Delivery of pRL-SV40 plasmid. MPG/DNA (black boxes) or MPGΔNLS/DNA (light grey boxes) complexes formed at a charge ratio of 5:1 with 100 ng pRL-SV40 plasmid encoding the reporter gene luciferase were overlaid onto synchronised or arrested (–FCS) human fibroblasts (HS-68). Luciferase activity was monitored 12 and 24 h after release from synchrony or after 24 h for arrested cells and 48 h for asynchronous cells. Control transfections were performed with Lipofectamine™ (white boxes) or naked plasmid (dark grey boxes).