Characterization of two nuclear localization signals in MSP58. A. Schematic diagrams of putative nuclear localization sequences (NLS1 and NLS2) in MSP58. Charged, basic amino acid residues are indicated in boldface. B. Schematic representation of the different EGFP-MSP58 fusion proteins tested and their localization pattern within cells. The point mutations were introduced at the two NLS loci by replacing the basic amino acid residues with alanine residues. The patterns were represented as being predominantly nuclear (N), nuclear and nucleolar (N + No), even throughout the entire cell (NC), cytoplasmic (Cyt), or nucleolar and cytoplasmic (No + Cyt). C. COS-1 cells were transfected with EGFP and EGFP-MSP58 mutant constructs as indicated in the figure and fixed 24 hours after transfection. The green color showed the fluorescence of the EGFP-fusion protein. Corresponding DAPI images are also shown. Total cell lysates from COS-1 transfected cells with the indicated constructs were analyzed by Western blotting using an anti-GFP antibody.