FIGURE 2.

Heterodimerization of PHOX2B polyalanine-expanded mutants with wild-type protein. A, representative immunoblotting images of co-immunoprecipitation of HA-tagged PHOX2B protein along with MYC-tagged PHOX2B polyalanine-expanded mutants 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). B, representative immunofluorescence images of the localization of the VP16 AD-PHOX2B +7Ala and VP16 AD-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 to the proteins detected by the anti-PHOX2B antibody (e and f). Scale bars, 10 μm. C and D, luciferase assays. The bars indicate the transcriptional activity of the pG5LUC reporter construct upon co-transfection in neuroblastoma SK-N-BE(2)C (C) or HeLa cells (D) with a vector containing the cDNA of wild-type protein fused to GAL4 BD (GAL4 BD-PHOX2B WT) in combination with the empty vector containing VP16 AD (hatched bars), the VP16 wild-type fusion protein (black bars), or the VP16-PHOX2B fusion protein carrying polyalanine expansions (cross-hatched 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 (C and D, n = 5) and are expressed as -fold increases over the activity of the reporter plasmid co-transfected with the GAL4 BD-PHOX2B WT protein (= 1). ***, significant differences from the activity of the wild-type protein fused to GAL4 BD (ANOVA, Tukey's test, p < 0.001).