Figure 5.

N50 peptide demonstrates specific binding to Imp α5 in a competition binding assay. Unlabeled N50 peptide (free N50) at indicated concentrations was incubated with whole cell lysate from Jurkat T cells. Biotinylated N50 peptide immobilized on agarose beads was added to pull down any importins not already complexed with free N50. Importins bound to beads were then quantified by immunoblotting. A, Immunoblots of importins pulled down from lysate total protein concentrations of 1.5, 0.3, and 0.05 mg/mL. Detection of importins in whole cell lysate is shown in the first column (lysate) for comparison to importins pulled down (beads). B, Quantitative representation of immunoblots from lysate total protein concentration of 1.5 mg/mL. The value of each importin bound to beads with no competition (0) was set to 100% and the percent of remaining importins bound after competition was calculated at each concentration of free N50 and shown as the mean±SD of 3 independent experiments. Please note that even high concentrations of free N50 peptide do not substantially inhibit binding of Imp α1 (open circles) or Imp α4 (open diamonds) to immobilized N50, but inhibition of Imp α5 (open triangles) binding to immobilized N50 is concentration dependent. Imp α indicates importin α; SD, standard deviation.