Fig. 10. Bpag1 contains a functional NLS in the plakin domain. (A) In both mouse and human Bpag1 proteins, the PVKRRRI/M motif in the plakin domain is conserved (identical amino acid residues have dark shading; similar amino acid residues have light shading). This motif is similar in the same region of mouse MACF, but differs in plectin (Plec1) and periplakin (Ppl). (B) The FLAG-Nterm2long plasmid was re-made with a mutation within the NLS sequence such that it would no longer resemble a classical NLS (Nterm2longNLS). The FLAG-NLS-ABD plasmid had the NLS coding sequence (coding for PVKRRRI) inserted immediately before the ABD insert. The mutant fusion proteins were of similar size to their wild-type counterparts (not shown). Labeling is the same as in Fig. 1. (C) Nterm2long fusion protein produced strong signal in the nucleus of 99% of the cells (arrowheads). The arrow in C points to some of the fusion protein co-aligning with stress fibers. (D) The Nterm2longNLS fusion protein staining never produced staining in the nucleus (arrowhead) above the background of untransfected cells. The protein was exclusively cytoplasmic, with much of it co-aligning with stress fibers (arrows). (E) Localization of the ABD fusion protein along stress fibers (arrow), but not in the nucleus (arrowhead). (F) With the NLS sequence placed before the ABD, 57% of the cells had labeling of the fusion protein within the nucleus (arrowhead), which was typically much stronger than the signal in the cytoplasm. The NLS-ABD fusion protein also co-aligned with stress fibers in many cells (arrow), and appeared no different than the ABD fusion protein in many. Bar in F, 20 µm, and also applies to CE. (G) Summary of the quantification of the localizations of the Nterm2long, Nterm2longNLS, ABD and NLS-ABD fusion proteins. Error bars indicate the standard errors from a minimum of three separate transfections.