ARTC2.2-Mediated Protein ADP-Ribosylation

In vitro modification of proteins from K562 cell extracts by ARTC2.2 recombinant protein was performed as described (Palazzo et al., 2016 (link)). Briefly, 8 × 10⁶ cells were washed twice in PBS and lysed in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X‐100, 0.2 mM DTT, 1 μM Olaparib, 4 mM Pefabloc SC PLUS (Sigma‐Aldrich) at 4°C. The cell extract was clarified by centrifugation and diluted five times in no-Triton X‐100 buffer and supplemented with 15 mM MgCl₂ and 1 μCi (37 kBq) of ³²P-NAD⁺. 67 μL extract aliquots were then incubated with or without 1 μM recombinant mARTC2.2 protein for 15 min at 30°C. After 15 min incubation, lysates were further incubated in presence of hydrolases for 45 min at 30°C. Samples were then analysed by SDS-PAGE and autoradiography.