Figure 3.

Amphioxus IGFBP Is Present in the Nucleus and Has Two Functional NLSs. A, Subcellular localization of amphioxus IGFBP. U2OS cells were transfected with the pC2-EGFP plasmid containing the indicated protein. eGFP signal was visualized (left panels) at 24 hours after transfection. Corresponding 4′,6-diamidino-2-phenylindole (DAPI) staining is shown in the middle panels and merged views in the right panels. Scale bar, 50 μm. B, Quantitative results of experiments described in panel A. In each experiment, more than 200 cells were counted and the percentage of cells showing nuclear eGFP signal was calculated and is shown. Values shown are means ± SEM; n = 3. ***, P < .001. C, Schematic diagrams of the amphioxus IGFBP/human IGFBP-2 chimeras tested. D, Nuclear localization of the chimeric proteins shown in panel C. Values are means ± SEM; n = 3. ***, P < .001. ns, not significant (P > .05). In the right panel, groups labeled with different letters are significantly different from each other (P < .05). E, Two NLS motifs are present in the L domain. Bold letters indicate basic residues. F, Subcellular localization of the indicated amphioxus IGFBP mutants. Values are means ± SEM; n = 3. Groups labeled with different letters are significantly different from each other (P < .05). WT, wild type.