FIGURE 6.

Analysis of subcellular localization of wild-type and mutant Oct4s. A, shown is immunocytochemical analysis of Tol2-Oct4-expressing ZHBTc4 clones. Cells were double-stained with anti-Nanog and anti-Oct4 antibodies. DAPI was used to visualize nuclei. Bar, 10 μm. B, shown is the effect of LMB on the subcellular localization of wild-type Oct4 and Oct4-NES-NES in stable ZHBTc4 clones. Cells were cultured in the presence of either EtOH (vehicle control) or LMB for 2 h, then fixed and stained with anti-Oct4 and anti-RanBP1 antibodies. The localization of endogenous RanBP1, an NES-containing protein known to shuttles between the nucleus and cytoplasm in a Crm1-dependent manner, was monitored to evaluate the effect of LMB. Mean fluorescence intensity of nuclear Oct4 staining for each sample is shown to the right. Data are the means ± S.D. The total number of analyzed nuclei (n) is as follows: WT, control (n = 89); WT, LMB (n = 119); NES-NES, control (n = 119); NES-NES, LMB (n = 112).