FIGURE 6.

Homodimerization of PHOX2B protein lacking the polyalanine tract and its heterodimerization with wild-type protein. A, representative immunofluorescence images of the localization of the GAL4 BD- and VP16 AD-PHOX2B ΔAla 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 of heterodimerization with the WT protein (B) or homodimerization (C) of PHOX2B ΔAla protein. The bars indicate the transcriptional activity of the pG5LUC reporter construct in HeLa cells upon co-transfection with a vector containing the cDNA of wild-type protein fused to GAL4 BD (GAL4 BD-PHOX2B WT; B) or a vector containing the cDNA of the deleted protein fused to GAL4 BD (GAL4 BD-PHOX2B ΔAla; 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 deleted of the polyalanine stretch (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 (n = 5) and are expressed as -fold increases over the activity of the reporter plasmid co-transfected with the GAL4 BD-PHOX2B WT protein (B) or GAL4 BD-PHOX2B ΔAla protein (C) (= 1). ***, significant differences from the activity of the wild-type protein fused to GAL4 BD (B) or the GAL4 BD-PHOX2B ΔAla (C) (ANOVA, Tukey's test, p < 0.001).