Fig. 3

The ADNP patient mutation impairs expression in the chromatin-enriched protein fraction. (A) 3D protein structure representation of the wild-type ADNP glutaredoxin active site (pink), NAP octapeptide sequence (fuchsia), eIF-4E interaction motif (blue), nuclear localization signal (dark cyan), homeobox domain (blue violet), and HP1 interaction motif (purple). The NAP domain (fuchsia) presents at the surface of the protein. (B) The nuclear localization signal-truncating p.His559Glnfs*3 mutant shows loss of the HP1-binding motif and DNA homeobox domain. (C) N-terminal ADNP detection in different subcellular compartments normalized to their protein fraction-specific loading controls. Detection of wild-type N-DYKDDDDK (Flag®)-tagged ADNP shows a molecular weight of 150 kDa. The p.His559Glnfs*3 mutant showed a lower molecular weight of 63 kDa. Cytoplasmic enrichment shows expression of wild-type ADNP (150 kDa) and the mislocalized p.His559Glnfs*3 mutant (63 kDa) with no difference in expression (p = 0.71; ns). Chromatin-enriched fraction demonstrated partial loss of mutant ADNP levels compared to wild-type ADNP, showing a dramatic decrease in expression (p = 0.03; *). Cytoskeletal fraction is enriched for wild-type ADNP and the p.His559Glnfs*3 mutant, with no significant difference in expression (p = 0.42; ns). GAPDH (cytoplasmic fraction), histone H3 (chromatin-bound fraction), and β-actin (cytoskeletal fraction) were used as loading controls. Statistical analysis of the subcellular fractionation immunoblots was performed using an unpaired two-tailed student T-test, assuming equal variances