Oded by a longer construct covering aa 21?62 was exclusively detected in the cytoplasm, indicating that the nuclear localization signal(s) residing within aa 172?25 are not operative in a more extensive protein context that includes the N-terminal domains (Figure 1B). With the exception of the construct encoding for aa 21?62 all YB-1 deletion constructs behaved similarly, indicating that for the tested model systems there are no major differences with regard to YB-1 protein targeting.Fine-mapping of nuclear localization signalsResultsSubcellular localization of YB-1 deletion constructsOur starting hypothesis was that YB-1 protein fragments may be directed to different cellular compartments. Analyses of the subcellular distribution for full-length and truncated YB-1-GFP fusion proteins has been described in HeLa cells [4]. We first confirmed these results in our model system. Fusion proteins encompassing either the full-length YB-1 or various deletions, possessing a C-terminal green fluorescent protein tag, were introduced into rat mesangial cells (RMC; Figure 1A). Some constructs encode for proteins with truncations of the C-terminal domain (denoted basic/acidic (B/A) or charged zipper domain); depicted in Figure 1A. To preserve comparability with previous results, we chose to introduce the same expression constructs used by Jurchott et al. [4]. Of note, the protein fragments span aa 1?17 of the YB-1 proteinTo narrow 2-Bromo-4-fluoro-5-methylbenzoic acid down the nuclear localization signals within the YB-1 protein, a computer-based search for known NLS was performed using the NUCDISC program (http://psort.nibb.ac.jp; [28]). The search revealed four hits, all residing within the C-terminal basic/acidic domain, that are (i.) aa 149?56, (ii.) 185?94, (iii.) 243?249 and (iv.) 276?92. These motifs were tested in isolation by fusing PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25386826 them to a DsRed fluorescent tag at the N-terminus. The subcellular localization was determined following expression of the respective fluorescent proteins in RMCs (Figure 2A, B). To readily visualize the cellular compartments a plasmid encoding for cyan fluorescent protein (CFP) was co-introduced. CFP is predominantly detected within the nucleus at 552 - 627 nm. CFP was chosen as the DsRed tag fluorescence spectrum overlaps with that of propidium iodine, thus precluding this method for nuclear counterstaining.van Roeyen et al. Cell Communication and Signaling 2013, 11:63 http://www.biosignaling.com/content/11/1/Page 3 ofThe residues encompassing aa 149?56, aa 185?94 and aa 276?92 conferred an exclusive nuclear fluorescence pattern, whereas the aa 243?49 motif did not (Figure 2A, B). Since the motif at aa 149?56, denoted PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 NLS-1, did not localize in the nucleus in the longer protein fragment encoded by P146-172 (Figure 1A, B), we therefore focused our attention on the motifs at residues aa 185?94 (NLS-2) and 276?92 (NLS-3) and evaluated their minimal composition for nuclear shuttling.Mutational analyses of the nuclear localization signals NLS-2 and -The NLS-2 at residue aa 185?94 has been described by Bader and Vogt in chicken YB-1 [29]. As a general rule, NLS are comprised of at least seven residues with a high content of basic amino acids [18]. Therefore we generated five different constructs by introducing mutations 2-Amino-3-methoxypyridine to narrow down the minimal requirement(s) for nuclear localization and specifically address the question whetherFigure 1 Localization of YB-1 and deletion constructs of YB-1. A. Schematic overview on GFP-tagged YB-1 (deletion) constructs.