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A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In <t>SEC–MALS</t> PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In <t>SEC–MALS</t> PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In <t>SEC–MALS</t> PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In <t>SEC–MALS</t> PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In <t>SEC–MALS</t> PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In <t>SEC–MALS</t> PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In <t>SEC–MALS</t> PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.

Journal: bioRxiv

Article Title: Dimerization of human PARP15 is required for NAD + binding and automodification

doi: 10.64898/2025.12.15.694324

Figure Lengend Snippet: A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.

Article Snippet: Light scattering detector Wyatt MiniDawn MALS and Wyatt Optilab refractive index (RI) detector were connected to a Shimadzu HPLC unit.

Techniques: Activity Assay, Mutagenesis, Hydrolysis Assay, Modification, Expressing, Incubation, In Vitro, Western Blot, Purification, Control