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. 2016 Apr 27;291(25):13375–13393. doi: 10.1074/jbc.M115.679027

FIGURE 4.

FIGURE 4.

Homodimerization of PHOX2A protein and its heterodimerization with PHOX2B wild-type protein and with polyalanine-expanded mutants. A and B, representative immunoblotting images of co-immunoprecipitation of MYC-tagged PHOX2A along with HA-tagged PHOX2A, PHOX2B (A), and PHOX2B polyalanine-expanded mutants (B) 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). H.E., higher exposure. C, luciferase assays. The bars indicate the transcriptional activity of the pG5LUC reporter construct upon co-transfection in HeLa cells with a vector containing the cDNA of wild-type protein fused to GAL4 BD (GAL4 BD-PHOX2B WT) in combination with the VP16 wild-type fusion protein (black bar) or the VP16-PHOX2A (cross-hatched bar); white, gray, and hatched bars indicate the transcriptional activity of the pG5LUC reporter construct upon co-transfection in HeLa cells with a vector containing the cDNA of PHOX2A fused to GAL4 BD (GAL4 BD-PHOX2A) in combination with the VP16-PHOX2A fusion protein (white bar), VP16-PHOX2B wild-type fusion protein (gray bar), or the VP16-PHOX2B fusion proteins carrying polyalanine expansions (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 (n = 4) and are expressed as -fold increases over the activity of the reporter plasmid co-transfected with the GAL4 BD-PHOX2B WT protein in combination with the VP16-PHOX2B wild-type fusion protein (= 1). ***, significant differences from the luciferase activity due to the wild-type protein homodimerization (ANOVA, Tukey's test, p < 0.001). D and E, luciferase assays. The bars indicate the transcriptional activity of the pG5LUC reporter construct upon co-transfection in HeLa cells with a vector containing the cDNA of PHOX2A fused to GAL4 BD (GAL4 BD-PHOX2A) in combination with the VP16-PHOX2A fusion protein (D, white bar) or the cDNA of wild-type protein fused to GAL4 BD (GAL4 BD-PHOX2B WT) in combination with the VP16 wild-type fusion protein (E, white bar). Black and hatched bars indicate the transcriptional activity of the pG5LUC reporter upon the co-transfection of the above plasmids with equimolar amounts of a vector encoding PHOX2B WT (black bars) or PHOX2B mutants (hatched bars). The results are the mean values ± S.D. (error bars) of the transcriptional activity of the constructs detected in at least three experiments performed in triplicate (D and E, n = 3) and are expressed as -fold increases over the activity of the reporter plasmid co-transfected with the GAL4 BD-PHOX2A protein (= 1; D) or the GAL4 BD-PHOX2B WT protein (= 1; E). ***, significant differences from the luciferase activity due to PHOX2B or PHOX2A homodimerization (ANOVA, Tukey's test, p < 0.001).