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Fig. 5. Essential structural components of Ngn1 and NeuroD required for the inhibitory actions on Nurr1-induced TH expression. (A-H) Effect of the abolishment of DNA-binding capacity of Ngn1 and NeuroD. Each of the wild types and the DNA-binding mutants of Ngn1 and NeuroD was introduced into Nurr1-transduced precursors by co-infection as described in the Materials and Methods, and TH+ cell numbers and TH protein levels were determined at day 3 of differentiation using immunocytochemical (A-G) and western blot analyses (H), respectively. Note that the abolishment of the DNA-binding capacity in Ngn1 and NeuroD abrogates not only their generic neurogenic activities (TuJ1 protein level in H), but also the inhibitory activity on Nurr1 and TH expression. (I) Schematic diagrams of the chimeric proteins of NeuroD (D) and Mash1 (M) or NeuroD and MyoD (Y) used to identify the domains for the differential roles of Mash1 (or MyoD) and NeuroD. Co-transduction of each of the chimeric proteins with Nurr1 was carried out as described above, and the effects of the chimeric bHLHs on Nurr1-TH expression were determined at day 3 of differentiation by immunochemical (J-R) and immunoblot (S) analyses. (J-S) Analyses of the chimeric proteins reveal that the HLH domains are responsible for the differentiation actions of Mash1 and NeuroD. Note that the effects of the chimera YD and MD, which hold NeuroD HLH domain, on Nurr1-induced TH+ cell numbers and TH protein levels are not distinguishable from those of wild-type NeuroD, whereas the inhibitory activity of NeuroD is abolished by substituting the HLH domain with that of Mash1 or MyoD (in chimera DM or DY). Graphs G and R represent the mean percentage (± s.e.m.) of TH+ cells out of total DAPI+ cells. *Significantly different from the controls at P<0.001. Bars, 10 µm.
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