Figure 4.

RanGTP is not sufficient to counteract the negative effect of importin β. Anchored chromatin-binding assays were analyzed by indirect immunofluorescence with anti-ELYS antibody as in Figure 1. Recombinant proteins were preincubated with cytosol before the addition onto chromatin-coated coverslips. (A) Representative images from binding reactions containing cytosol supplemented with 20 μM ovalbumin (control), 20 μM Xenopus importin β (XImp β), or 20 μM Xenopus importin β + 40 μM RanQ69L-GTP (XImp β + RanGTP). Scale bar, 10 μm. (B) Quantitative analysis summarizing three separate experiments of the type shown in A. Anti-ELYS immunofluorescent signal intensity was measured exclusively from the chromatin surface on 12 randomly chosen, nonoverlapping templates in each category. Normalized fluorescence intensity is shown after the subtraction of nonspecific staining, measured on identical coverslips for which the primary antibody was omitted from the procedure. Error bar, SD.