Characterization of the (A) PNKP and (B) ATXN3 immunocomplexes by Western blot analysis. HEK-293 cells were transfected with either PNKP siRNA or ATXN3 siRNA or control siRNA and the nuclear extracts (1 mg) prepared from those cells were IP’d with anti-PNKP Ab (BioBharati Life Science Pvt. Ltd, Kolkata, India; 2A) or anti-ATXN3 Ab (Proteintech, 2B) or with IgG as a control, and tested for the presence of PNKP- and ATXN3- associated proteins with Abs to the proteins shown on the right. (C) Detection of PNKP’s interaction with ATXN3 in SH-SY5Y cells by proximity ligation assays using a Duolink kit (Olink Bioscience, Uppsala, Sweden). Nuclei were counterstained with DAPI (blue). ATXN3-depleted (by siRNA) cells were used as a control to show the specificity of the interaction (middle panel). Right panel, Non-specific Ab (IgG) control (D) GST-PNKP pull-down of ATXN3 (WT and mutant) using purified GST-tagged full-length or three domains (FHA-, Phosphatase- and Kinase-domain) of PNKP, probed with anti-ATXN3 antibody. Bottom panel, Coomassie-stained gel of the corresponding PNKP domains, a second gel run in parallel. A replicate lane, showing full length purified PNKP (between lanes 2 and 3), was cut to avoid repetition and the remaining gel sections were joined together (indicated by a black solid line). The spliced sections originated from the same gel.

https://doi.org/10.1371/journal.pgen.1011124.g001