In vitro PARylation and Phosphorylation Assays

In vitro PARylation assays were performed as described previously (Kong et al., 2021 (link); Yao et al., 2021 (link)). Briefly, 500 ng HIS-FonPARP1 were incubated in a 50 μL PARylation buffer (50 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl₂, pH8.0) with 0.2 mM NAD⁺, 1× activated DNA (BPS Bioscience, San Diego, CA, USA), and 3 μg GST-FonKin4 at 26°C for 3 h. Subsequently, the samples were separated on 8% and 12.5% SDS-PAGE. PARylated proteins were detected via immunoblotting with anti-poly-ADPR antibody (Cat. #MABE1031, Sigma-Aldrich), anti-GST antibody (Cat. #A00865, GenScript) or anti-HIS antibody (Cat. #A00186, GenScript).
For in vitro phosphorylation-mediated self-PARylation assays, 4 μg HIS-FonPARP1 underwent prior in vitro phosphorylation by HIS-FonKin4-ST, as described above. Then, 50 μL PARylation buffer was then added, followed by incubation at 26°C for 30 min. The reaction was stopped by adding 4× SDS loading buffer, and the proteins were then separated on 8% and 12.5% SDS-PAGE. Phosphorylated proteins were detected by anti-phosphor Ser/Thr antibody. PARylated proteins detected through immunoblotting with streptavidin-HRP (Cat. #21126, Thermo Fisher Scientific, Waltham, MA, USA) for Biotin NAD⁺ or anti-HIS antibody (Cat. #A00186, GenScript).