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. 2016 Dec 20;11(12):e0168617. doi: 10.1371/journal.pone.0168617

Fig 3. Interaction of DdNKAP and Prp19.

Fig 3

(A) Schematic representation of the Prp19 domain structure and Prp19 constructs used in this study. (B, C, D, E) GST tagged full length Prp19, the Prp19-like and the 134–264 domains pull down GFP-DdNKAP and GFP-DdBasic recognized by mAb K184-3. GST-Prp19 polypeptides and GST were visualized by Ponceau S staining. Proteins were separated by SDS-PAGE (12% acrylamide). The GFP-DdBasic domain interacts with GST-Prp19, GST-Prp19-like and GST-134-264. (F, G, H) The GST tagged full length Prp19 and truncated Prp19 proteins were incubated with bacterially expressed, purified and thrombin cleaved DdRS (F), DdBasic (G) and DdDUF (H). Beads were washed and proteins were separated by SDS-PAGE (15% acrylamide) and visualized by Coomassie Blue staining. DdRS, DdBasic and DdDUF are marked with red asterisks; the red box denotes the direct interaction of DdBasic with GST tagged full length Prp19, Prp19-like and 134–264. (I) The DdRS domain carries several predicted potential ubiquitination sites (indicated in red). GFP and GFP-DdNKAP were immunoprecipitated by GFP-Trap beads. The ubiquitinated DdNKAP was detected by western blot with anti-ubiquitin P4D1 mAb and the immunoprecipitation of GFP-DdNKAP and GFP was monitored by mAb K3-184-2.