Fig. 7.

Schematic overview of Poly-ADP-Ribose Assisted Protein Localization AssaY (PARAPLAY). a To generate a construct encompassing the protein of interest in fusion with PARP1cd an expression vector (left) can be used harboring a multiple cloning site (MCS) to insert the cDNA encoding the protein of interest upstream of the PARP1cd cDNA. An optional C-terminal tag for protein detection may be included. Transfection of cells with the resultant vector leads to robust PAR accumulation, if the overexpressed protein is targeted to an organellar lumen (right). b Co-transfection of cells with the PARP1cd fusion construct and an organelle-targeted PARG may be conducted to confirm the results. PARG cleaves PAR to ADP ribose monomers, which are not detectable by PAR immunochemistry. Therefore, if the PARP1cd fusion protein resides within the same organelle as the targeted PARG, PAR accumulation is not detectable any more