Fig. 4. Subcellular localization and heterodimer formation with E47. (A) Flag-tagged neurogenic differentiation 1 (NeuroD1) wild type (WT) and mutants were expressed in HEK 293T cells and subcellular proteins were fractionated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), LaminB, or Histone H3 were used as internal controls for the cytoplasmic extract (CE), nuclear extract (NE), and chromatin bound extract (ChE). (B) Data from (a) are represented as means±S.E. from 3 independent experiments (#p<0.05, compared to the value of the WT CE; *p<0.05; **p<0.01, compared to the value of WT NE). (C) Immunocytochemistry showing Flag-tagged WT, S274A, and S274D in HEK 293T cells. Flag (red); green fluorescent protein (GFP) (green); Hoechst (blue) for nuclei. (D) The fluorescence intensity of Flag-tagged NeuroD1/GFP was quantified using the ImageJ software program. Data from (c) are represented as means±S.E. with respect to the value of WT (*p<0.05). (E) Co-immunoprecipitation assay was used to assess heterodimer formation of NeuroD1 and E47. Note that the WT, S274A, and S274D retain similar binding activity to E47 protein. Actin was used as a loading control.
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