p35 interacts with NIF-1.

<p>(A) Mouse NIF-1 encodes a 1,291-amino acid protein and contains 6 zinc finger domains, an LXXLL domain, and a leucine zipper-like motif. (B & C) Mapping of interaction domains between NIF-1 and p35. Yeast was co-transformed with different domains of p35 and NIF-1. +, strong interaction; −, absence of interaction. (B) The C-terminal region of NIF-1 (amino acids 1,066–1,291) containing the 6^th zinc finger domain, leucine zipper-like motif, and short C-terminus was sufficient to interact with p35. (C) The N-terminal region of p35 (corresponding to the p10 fragment) was required for the interaction between p35 and NIF-1. p10 comprises the 98 N-terminal amino acids of p35, while p25 contains the C-terminal region of p35. (D & E) Direct interaction between NIF-1 and p35. Recombinant GST fusion proteins encoding different regions of NIF-1 were incubated with lysate prepared from p35-overexpressing COS-7 cells (D) or recombinant p35 protein (E). The bound proteins were pulled down by glutathione-Sepharose and analyzed by western blot analysis (Lysate, as an input control). Bottom panel: Coomassie-stained gel. (F) Association of p35 with NIF-1 in mammalian cells. COS-7 cells were transiently transfected with NIF-1 and p35. Cell lysate was immunoprecipitated (IP) with p35 or NIF-1 antibody as indicated and subjected to western blot analysis. Rabbit normal IgG (IgG) was used as a negative control.</p

p35 regulates NIF-1 subcellular localization.

<p>(A) Nuclear localization of NIF-1 in COS-7 cells. HA-tagged NIF-1 expressing COS-7 cells were stained with HA and NIF-1 antibody. The nuclei were stained with DAPI. The specificity of the NIF-1 antibody was confirmed by pre-absorption of the antibody with the immunogen (HIS-tagged NIF-1 fusion protein; bottom panels). (B) Cellular distribution of p35 and NIF-1 in COS-7 cells. p35 or HA-tagged NIF-1 expressing COS-7 cells were stained with HA and p35 antibodies, respectively. The specificity of p35 and NIF-1 staining was confirmed by the negative staining signals of the neighboring non-transfected cells (C) The expression of p35 but not p39 abolished the exclusive nuclear accumulation of NIF-1. HA-tagged NIF-1 was co-expressed with p35 or p39 in COS-7 cells. NIF-1 was stained with HA antibody, and p35 and p39 were stained with their corresponding antibodies. (D) COS-7 cells were transfected as described in B and stained with NIF-1 antibody followed by Alexa Fluor 488-conjugated anti-rabbit IgG. Micrographs are representative images of transfected cells. (E) Quantitative analysis of the cells that exhibited exclusive nuclear accumulation of NIF-1. Cells (<i>n</i> = 100) were scored for each condition. Results represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, significantly different from that of the cells expressing NIF-1 alone, one-way ANOVA followed by the Student–Newman–Keuls test). Scale bar = 10 µm.</p

Dependence of p35 nuclear export on its NES.

<p>(A) Consensus sequence of the NES on p35. The functional NES comprises a core of closely spaced leucine residues or other hydrophobic amino acids. The critical hydrophobic residues in the putative NES of p35 are underlined. An NES mutant of p35, p35NES, was generated by mutating the 3 conserved hydrophobic residues (i.e., leucine 226, 227, and 230) to alanine. (B) The NES of p35 is required to mediate the nuclear export of NIF-1. COS-7 cells were co-transfected with HA-NIF-1 and p35WT or p35NES and subsequently stained with HA antibody. The results of the quantitative analysis represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, one-way ANOVA followed by the Student–Newman–Keuls test). (C) Mutating the NES of p35 increased the population of Neuro-2A cells containing nuclear p35. Neuro-2A cells were transfected with p35WT-GFP or p35NES-GFP and subsequently differentiated by RA. The cells were stained with p35 antibody. Representative fluorescent images depicting the localization of p35WT and p35NES (indicated by the p35 staining and GFP expression). (D) Quantitative analysis of cells with nuclear p35. Cells with nuclear p35 were counted if the GFP signal in the nucleus was>75% than that of the cytoplasm. Results represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, Student's <i>t</i>-test). (E) LMB treatment caused the accumulation of p35 in the nucleus of cortical neurons. Cultured cortical neurons were treated with LMB for 1 h, and subcellular fractionation was performed. Western blot analysis of p35 and Cdk5. Total: protein extracted from the same batch of neurons using RIPA.</p

Mediation of p35-triggered NIF-1 nuclear export via a CRM1-dependent pathway.

<p>(A & B) COS-7 cells were transfected with p35 and HA-tagged NIF-1. Twenty-four hours after transfection, the cells were treated with LMB (5 or 10 ng/mL) for 4–8 h, stained with HA antibody, and examined by fluorescence microscopy. Representative fluorescence images (A) and quantitative analysis of the cells showing exclusive nuclear accumulation of NIF-1 (B). At least 100 cells were scored for each condition in each trial. Results represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, one-way ANOVA followed by the Student–Newman–Keuls test). (C–E) p35-stimulated redistribution of NIF-1 from the nucleus to cytoplasm is independent of Cdk5 activity. (C) Active Cdk5 phosphorylated recombinant NIF-1 protein. Recombinant GST-NIF-1-C protein was subjected to phosphorylation assay by Cdk5 (GST and histone H1 were used as negative and positive controls, respectively). (D) COS-7 cells were transfected with the HA-tagged NIF-1 and p35, and subsequently treated with roscovitine (Ros, 10 or 25 µM) for 4 or 8 h. The cells with exclusive nuclear accumulation of NIF-1 were quantified. (E) Inhibition of Cdk5 activity by dnCdk5 expression did not affect the p35-stimulated nuclear export of NIF-1. COS-7 cells were transfected with NIF-1, p35, and dnCdk5 constructs as indicated. Quantitative analysis as described in (D). Results represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, one-way ANOVA followed by the Student–Newman–Keuls test).</p