PNKP interacts with both wild-type and mutant ATXN3 in cells and human brain sections.

<p><b>(A)</b> Plasmids pGFP-ATXN3-Q28 and pCherry-PNKP were co-transfected into SH-SY5Y cells and co-localization of PNKP and ATXN3-Q28 was assessed by confocal microscopy; the merge of green and red fluorescence from ATXN2-Q28 and PNKP, respectively, appears as yellow/orange fluorescence. Nuclei were stained with DAPI in C-D. <b>(B)</b> Plasmids pGFP-ATXN3-Q84 and pCherry-PNKP were co-transfected into SH-SY5Y cells and co-localization of PNKP and ATXN3-Q84 was assessed as in A. <b>(C)</b> Bimolecular fluorescence complementation assay. SH-SY5Y cells were transfected with plasmid: Panel 1) pVN173-PNKP; Panel 2) pVC155-ATXN3-Q28; Panel 3) pVC155-ATXN3-Q84; Panel 4) co-transfected with pVN173-PNKP and pVC155-ATXN3-Q28; or Panel 5) pVN173-PNKP and pVC155-ATXN3-Q84. Reconstitution of green/yellow fluorescence was assessed 48 hours after transfection using fluorescence microscopy (20X). Nuclei were stained with DAPI in Figs. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004834#pgen.1004834.g001" target="_blank">1C and 1D</a>. <b>(D)</b> Proximity ligation assays (PLAs) were performed on control and SCA3 human brain sections, using mouse anti-PNKP and rabbit anti-ATXN3 primary antibodies, and anti-DNA ligase 3 (DNA LIG3) (rabbit) with anti-ATXN3 (mouse) antibodies as control. The generation of red fluorescence was monitored under a fluorescence microscope.