Figure 4

ASV IN NLS import does not compete for import factors required for SV40-TAg and U1A nuclear accumulation. A. Digitonin permeabilized HeLa cells were either treated with buffer (PBS – top row), or with a molar excess of the competitor protein trxIN(195–270) (bottom row). GST-IN(1–286) and GST-TAg had a 15-fold excess of competitor while GST-IN-NLS(201–236) had a 30-fold molar excess. Import assays were performed as shown in Fig. 3 and staining was done with fluorescent antibody against GST. B. Quantitative analysis of nuclear import of various GST fusion proteins with (+ comp) and without (no comp) competitor. More than 100 cells were counted for each experimental condition and the percentage of cells that had a mostly nuclear staining for the fusion protein was calculated. The percent decrease in the presence of the competitor is shown in the column on the right. The lower value for import of GST-IN (201–236) compared to GST-IN (1–286) reflects the fact that a larger percentage of cells had whole cell staining (in which nuclear import could not be assessed) or nuclear exclusion. C. Digitonin permeabilized HeLa cells were either treated with buffer (PBS – top row), or with a 50 ug/ml antibody 3E9 against Impβ (bottom row)during the import reaction. D. Quantitative analysis of nuclear import of various GST fusion proteins with (+ Ab3E9) and without (no Ab) antibody 3E9. More than 100 cells were counted for each experimental condition and the percentage of cells that had a mostly nuclear staining for the fusion protein was calculated. The percent decrease in the presence of the antibody is shown in the column on the right.