FIGURE 1.

Homodimerization of PHOX2B wild-type protein. A, representative immunoblotting images of co-immunoprecipitation of MYC- and HA-tagged PHOX2B proteins in transfected HeLa cells. Cell extracts were immunoprecipitated with anti-MYC antibody or control immunoglobulin (IgG) and immunoblotted with anti-MYC (top) and anti-HA antibodies (bottom). B, representative immunofluorescence images of the localization of the GAL4 BD- and VP16 AD-tagged PHOX2B WT 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. 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 combination of equimolar amounts of a vector containing the cDNA of wild-type protein fused to GAL4 BD (GAL4 BD-PHOX2B WT) and the empty vector containing VP16 AD (hatched bars) or with a combination of equimolar amounts of both GAL4 BD and VP16 wild-type fusion proteins (black bars). Nonspecific interactions were excluded upon co-transfection of PHOX2B fused to VP16 AD with VP16 AD or GAL4 BD (light and dark gray bars). The background level of firefly luciferase expression from the pG5LUC vector was determined upon co-transfection with empty vectors containing GAL4 BD and VP16 AD (white 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 < 0.05); ***, significant differences from the activity of the wild-type protein fused to GAL4 BD (ANOVA, Tukey's test, p < 0.001).