Basic residues within each of the NLSs are required for nuclear localization of PK. (A) Schematic representing the three different NLSs bearing neutralizing mutations in the basic residues were cloned in-frame with the cytoplasmic protein PK bearing a c-Myc tag. Results demonstrated in (B-D) are summarized in the column to the right, where "Np" designates nucleoplasmic localization and "C" designates cytoplasmic localization. (BD) Subcellular localization was determined by immunofluorescence in HeLa cells using an α-myc primary and either a HRP or Cy-3 conjugated secondary antibody. Costaining of the nucleus with DAPI is shown in blue. (B) Mutations in either the first (NLS1-sub1), second (NLS1-sub2), or third (NLS1-sub3) basic cluster of NLS1 were mutated in alanine and visualized for nuclear localization. (C) Expression and localization of NLS2 mutants lacking either the first (NLS2-sub1), second (NLS2-sub2), or third (NLS2-sub3) basic cluster. (D) Expression and localization of the constructs bearing mutations in the first (NLS3-sub1) or second (NLS3-sub2) basic cluster of NLS3.