Figure 2

PARP3 interacts with and ADP ribosylates G9. (a–c) G9a, GLP1 and Wiz co-immunoprecipitate with Flag-PARP3^WT and Flag-PARP3^HE but not with Flag. The PARP3^−/−3-Flag, PARP3^−/−3-Flag-PARP3^WT and PARP3^−/−3-Flag-PARP3^HE LN229 cell extracts were immunoprecipitated with a Flag antibody and analysed by western blotting using anti-G9a (a), anti-GLP1 (b), anti-Wiz (c) and anti-Flag antibodies. Input corresponds to 1/40 of the total amount of cell extracts used for immunoprecipitation. (d) The absence of PARP3 does not alter the stability of the G9a/GLP1/Wiz complex. The parental (WT) and the PARP3^−/−3 LN229 cells were transfected with either EGFP (control) or EGFP-G9a. EGFP and EGFP-G9a immunoprecipitates were analysed by western blotting using anti-Wiz, anti-GLP1 and anti-EGFP antibodies. Input corresponds to 1/30 of the total amount of cell extracts used for immunoprecipitation. (e) PARP3 ADP-ribosylates G9a in vitro. Immunopurified EGFP (control) or EGFP-G9a were incubated with purified PARP3 in the absence or in the presence of DNase-activated DNA and biotinylated NAD⁺. When indicated ME0328 (20 µM) was added in the reaction buffer to inhibit PARP3 catalytic activity. ADP-ribosylated G9a and PARP3 (auto-ADP-ribosylated) are detected using the streptavidin Alexa system. EGFP and EGFP-G9a are detected by western blotting using an anti-GFP antibody. Uncropped Western-blots are shown in the supplementary information file.