SH-SY5Y cells immunolabeled with SMI-31 mAB or RT-97 mAB demonstrated discrete nuclear localizations in nuclei with pronounced staining in neuronal processes and diffuse staining within cell bodies. Confocal microscopy demonstrated clearly that the antibodies label structures within the nucleus. Comparison among antibodies that are specific for phosphorylated IFs (SMI-31, RT-97) and those not (SMI-32, anti-GFAP) suggests that the nuclear structures are a phosphorylated IF. Cross reactivity of both SMI-31 and RT-97 on glial cytoplasm and nuclei indicates the epitope is not cell type specific. Examination of western blots indicate SMI-31 and RT-97 recognize, among others, a protein in the 66 – 74 kDa range in extracts of both neuroblastoma and glioma cells. Western analysis with anti-lamin B labeled a similar band in the same extracts. The calculated molecular weights of lamins A, B and C are approximately 74, 66, and 65 kDa, respectively; therefore, it seems most likely that lamins, NF-H, and GFAP all share a common motif that, when phosphorylated, is recognized by SMI-31 and RT-97 mABs. In support of this, a search for phosphorylation motifs among NF-H, GFAP and lamins A, B, and C revealed a single, high probability tyrosine site that occurs once on each of these proteins (Figure 3), further suggesting the possibility that this site constitutes or is contained within the epitope recognized by these two antibodies.
Based on the finding of a shared, tyrosine phosphorylation site, it seems likely that the nuclear structures reported in this and previous studies [1, 12] that employed SMI-31 are phosphorylated lamins. Furthermore, nuclear labeling and DNA synthesis appeared tightly associated in SH-SY5Y cultures, consistent with the suspected role of lamins in DNA replication . Others have reported SMI-31 labeling in glial as well as neuronal nuclei. For example, Schilling et al.  identified developmentally regulated nuclear antigens recognized by SMI-31 mAB in C6 rat glioma cells in vitro as well as glial and neuronal nuclei in vivo, suggesting the presence non-neurofilament epitopes. In addition, Shea et al. , in a study of neurite outgrowth from NB2a cells, showed that both the SMI-31 and RT-97 mABs strongly labeled nuclei of undifferentiated cells, and the nuclear labeling declined as differentiation progressed. These results are similar to the results reported here. In a recent paper by Glass et al. , SMI-31 was described as labeling nuclear epitopes in cells in culture and in neurons of mature rat brain as visualized by cryo-sectioning and confocal microscopy. However, in light of the findings reported here, the nuclear objects reported by Glass et al. in rat brain sections may have been in mitotically active glial cells as well as in neurons.
Although these nuclear epitopes appear similar to nuclear speckles, the latter have not been reported as IF-associated structures, but rather storage sites of RNA processing proteins (see Sanford 2002, for a current, brief review ). However, as it is know that lamins are involved in nucleo-skeletal organization  and organization of DNA replication sites , it would not be unexpected to find lamins associated with nuclear speckles, perhaps as structural organizers.