Fig. 4.

Anhydrin reduces aggregation of EGFP-PABPN1-A17 in mammalian cell nuclei. (A–H) Confocal microscopy of T-REx293 cells expressing EGFP-PABPN1-A17 (B, D, F, H) with mCherry (C, D) or anhydrin-mCherry (G, H). Nuclei expressing EGFP-PABPN1-A17 are green (white arrowheads indicate aggregates and yellow arrowheads normal nuclear speckles), cells/nuclei expressing mCherry or anhydrin-mCherry are red and nuclei are stained blue with DAPI (A, D, E, H). (I) Western blot of cytoplasmic and nuclear fractions of cells transfected with either empty pCMVFLAG5a vector or His₆-anhydrin-Flag construct and probed with antibodies against Flag (top), histone H3 (middle) or tubulin (bottom). (J and K) Percentage of nuclei with EGFP-PABPN1-A17 aggregates after either mCherry (black) or anhydrin-mCherry (gray) coexpression; in K, cells were treated with the proteosomal inhibitor lactacystin; results show SD and are highly significant by logistic regression analysis. ***, p < 0.001. (L) Example FRET analysis of EGFP-PABPN1-A17 (donor)/anhydrin-mCherry (acceptor) interactions in a live cell, showing DIC transmission image (tm), signal in donor channel upon excitation at donor wavelength (dx/dm), signal in acceptor channel upon excitation at acceptor wavelength (ax/am), and donor normalized and unmixed FRET transfer efficiency dFRET (labeled FRET).