HPF1, PARP2 and Nuc165 form an enzymatically active complex in solution.

A: Electrophoretic mobility shift assay of Nuc165 (1 μM), Nuc165 (1 μM) + 2 molar equivalents of PARP2-WT_Alexa488 (2 μM), and Nuc165 (1 μM) + 2 molar equivalents of PARP2-WT_Alexa488 (2 μM) + 10 molar equivalents of HPF1_Alexa647 (10 μM). The nucleosome was detected by SYBR Gold staining, PARP2-WT_Alexa488 and HPF1_Alexa647 were detected by their Alexa 488 and Alexa 647 fluorescence emission, respectively (right panels). B: Unnormalized size exclusion chromatograms shown in Fig 4B and SDS-PAGE analysis of collected fractions. Blue traces represent absorbance at 280 nm, red traces represent absorbance at 260 nm. Elution volumes are indicated for each peak. C: Uncropped autoradiogram shown in Fig 4D. ADP-ribosylation reaction by PARP2-WT, PARP2-QFRD (same data as in Fig 4D) and PARP1 of a peptide of H31-21, in absence and presence of HPF1. D: Uncropped autoradiogram shown in Fig 4E. ADP-ribosylation reaction by PARP2-WT, PARP2-QFRD (same data as in Fig 4E) and PARP1 of histones in Nuc165, in absence and presence of HPF1. Free 32P-NAD+ was run alone in the last lane as a control. (RAR)