Detection of PARP-7 autoMARylation by western blotting

The autoADPRylation reaction described above was stopped by the addition of one third of a reaction volume of 4x SDS-PAGE Loading Buffer (200 mM Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 4% β-mercaptoethanol, 50 mM EDTA, 0.08% bromophenol blue) followed by incubation at 100°C for 10 min. The reactions were then loaded onto a 10% PAGE-SDS gel and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk in TBST, the membrane was then blotted with antibodies against PARP-7 (Invitrogen) or Flag (Invitrogen), or an ADP-ribose detection reagent (MABE1016, EMD Millipore), followed by blotting with anti-rabbit HRP-conjugated IgG (1:5000) or anti-mouse HRP-conjugated IgG (1:5000) secondary antibodies. Immunoblot signals were detected using an ECL detection reagent (Thermo Fisher Scientific, 34577, 34095), and quantified using Image Lab 6.0 (Bio-Rad).