Figure 1

Subcellular localization of Phox2B variants in SH-SY5Y cells. (A) The wild-type (20Ala) variant of Phox2B is present almost exclusively in the nucleus, consistent with the protein’s function as a transcription factor. The addition of further alanine residues, representing milder and later-onset versions of CCHS (23-30Ala), are also primarily present in the nucleus. Only the most significant alanine expansion, 33Ala, contains misfolded Phox2B in the cytosol. Nuclear imaging with Hoechst staining and Phox2B via an mCherry N-terminal tag; (B) Consistent with previous attempts to express the most significant clinical versions of Phox2B, the addition of 13 extra-alanine residues results in the cytoplasmic localization of the protein in ~50% of cells. At baseline, no other Phox2B variant had a significant number of cells with misfolded, cytoplasmically localized protein (all less than 5%, including the WT protein). Data are presented as mean ± s.e.m. of biological triplicate experiments, where p < 0.05 (denoted by*) was determined using a one-way ANOVA with a Dunnett’s post-hoc test relative to the control (WT) group.