Fig. 2: Exploiting PARP15.1 localization for assay development.
From: Screening assay to monitor mono-ADP-ribosylhydrolase activity of viral macrodomains in cells
a Schematic representation of the assay illustrating foci formation of MARylated PARP15.1, which is reversed upon hydrolase activity of a macrodomain (created in BioRender. Verheugd, P. (2026) https://BioRender.com/zm1a7k2). b Schematic representation of constructs designed to generate cell lines stably expressing the indicated fusion proteins. The integration of a T2A site between GFP-PARP15 and the 3xFLAG-NLS-NBGFP (a GFP-specific nanobody) enables the simultaneous expression of both fusion proteins in equimolar ratios. Additionally, the inclusion of a Gateway cassette downstream of the sequence encoding the NBGFP facilitates recombination of various macrodomains or their respective mutants for analysis. GFP green fluorescent protein, MD macrodomain, NLS nuclear localization signal, NB nanobody. c The indicated constructs were transiently expressed in HEK293 cells. Protein expression was analyzed by immunoblotting. GFP-PARP15.1 was detected using a specific PARP15 antibody, 3x-FLAG-NLS-NBGFP fusion proteins were visualized by an anti-FLAG antibody. γ-Tubulin served as loading control. d U2OS cells were transiently transfected with constructs as shown in (b) expressing GFP-PARP15.1 and the indicated NBGFP, either without the MD, with the SARS-CoV-2 MD, the CHIKV MD, or its catalytically inactive mutant (V33E). Cells were fixed, nuclei visualized by Hoechst staining (blue) and localization of GFP-PARP15.1 (green) and 3xFLAG-NLS-NBGFP variants (red) determined by confocal microscopy.
