FIGURE 3.

Homodimerization of PHOX2B polyalanine-expanded mutants. A, representative immunofluorescence images of the localization of the GAL4 BD-PHOX2B +7Ala and GAL4 BD-PHOX2B +13Ala fusion proteins in transfected HeLa cells stained using anti-PHOX2B antibody (c and d). The nuclei were visualized using DAPI (a and b) and merged with the proteins detected by the anti-PHOX2B antibody (e and f). Scale bars, 10 μm. B and C, luciferase assays. The bars indicate the transcriptional activity of the pG5LUC reporter construct upon co-transfection in neuroblastoma SK-N-BE(2)C cells with a vector containing the cDNA of PHOX2B +7Ala fused to GAL4 BD (GAL4 BD-PHOX2B +7Ala in B) or the cDNA of PHOX2B +13Ala fused to GAL4 BD (GAL4 BD-PHOX2B +13Ala in C), in combination with the empty vector containing VP16 AD (hatched bars), VP16 wild-type fusion protein (cross-hatched bars), or VP16-PHOX2B fusion protein carrying +7 (Fig. 3B) or +13 (Fig. 3C) alanine expansions (black bars). The results are the means ± S.D. (error bars) of the transcriptional activity of the constructs detected in at least three experiments performed in triplicate (B and C, n = 4) and are expressed as -fold increases over the activity of the reporter plasmid co-transfected with the GAL4 BD-PHOX2B +7Ala protein or the GAL4 BD-PHOX2B +13Ala protein (= 1). ***, significant differences from the activity of the PHOX2B protein +7 or +13 alanine fused to GAL4 BD (ANOVA, Tukey's test, p < 0.001). D, representative immunoblotting images of co-immunoprecipitation of HA-tagged PHOX2B polyalanine-expanded mutants along with MYC-tagged PHOX2B wild-type and mutant variants in transfected HeLa cells. Cell extracts were immunoprecipitated with anti-HA antibody or control immunoglobulin (IgG) and immunoblotted with anti-HA (top) and anti-MYC antibodies (bottom).