EZH2 interacts with PARP1 and is PARylated after DNA damage induction (A) Immunoprecipitation of EZH2 with PARP1 under physiological conditions and after induction of DNA damage. His-PARP1 was expressed in HeLa cells by transfection. Cells were treated with 100 uM of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 10 minutes to induce DNA damage and activate PARP1. Input corresponds to 1/20 th of protein extracts from transfected cells used for the tag-construct pulldown. (B) Proteins interacting with EZH2 were analyzed by His pulldown or immunoprecipitated with non-immunogenic IgG (control) followed by western blot analysis with anti-EZH2 (top), anti-PAR (middle) and anti-PARP1 (bottom) antibodies. (C) Immunoprecipitation of EZH2 with PAR-affinity resin after induction of DNA damage. LCLs and HeLa cells were treated with or without 100 uM MNNG for 10 minutes. Cellular protein extracts were immunoprecipitated with a PAR affinity resin or PAR negative control resin and analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. Input corresponds to 1/10 th the amount of cell extracts used for immunoprecipitation.