Fig. 1.

Classic MBP constructs, subcellular targeting, and proliferation assays. A: Schematic diagram of the RFP-tagged 18.5-kDa and 21.5-kDa MBP constructs, and their complementary reverse localization variants, that were used throughout this study to distinguish whether the nuclear localization of MBP, or the isoform specificity, was required for phenotypic gain of function. The inclusion of exon II in the 21.5-kDa isoform and the minimal 21-nt 3′-untranslated region (UTR) are also shown for completeness. B: Fluorescence micrographs of cultured N19-OLGs 3 days posttransfection expressing 18.5-kDa or 21.5-kDa MBP isoforms and their complementary reverse localization variants (red). The 2xNLS signal added as a linker between RFP and 18.5-kDa MBP traffics the protein to the nucleus, whereas the 2xNES added to 21.5-kDa MBP exports the protein out of the nucleus to the cytoplasm and plasma membrane. C: Cultured N19-OLGs expressing the four MBP constructs were processed for BrdU incorporation following a 24-hr BrdU pulse (BrdU administered between 48 hr and 72 hr posttransfection). The BrdU was detected using immunohistochemical diaminobenzidine staining. D: The percentage of total cells incorporating BrdU was determined by a blinded observer counting 10 random fields in three different experiments. A statistically significant increase in number of cells incorporating BrdU of 13% (**P = 0.05) was observed for RFP-MBP-21.5, and no increases in cell proliferation were seen in cultures transfected with either RFP-MBP-18.5 or RFP-2xNLS-MBP-18.5. Cell cultures transfected with RFP-2xNES-MBP-21.5 did not show any increases in cell proliferation, supporting the conclusion that the nuclear localization of 21.5-kDa MBP is required for cell proliferation. The 18.5-kDa MBP isoform, when localized to the nucleus, also did not increase the rate of cell proliferation, suggesting that this process requires the presence of the exon II-encoded segment. A color version of this figure can be found in the online version of this article. Scale bars = 20 μm in row 1; 50 μm in rows 2, 3. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]