Figure 6. TIPARP mono-ADP-ribosylates AHR in vitro.

(A,B) One microgram of GST-tagged TIPARP protein was incubated with 2 µg of GST-AHR 430–848 and 2 µCi ³²P-NAD⁺. Mono-ADP-ribosylation of proteins was detected by autoradiography after SDS–PAGE and levels of proteins loaded were visualized by GCB staining. Repression of TCDD-induced CYP1B1-luc reporter gene activity by (C) TIPARP N-terminal truncations, (D) TIPARP zinc-finger (C243A) and catalytic mutant (H532A) point mutants. Full-length TIPARP (1–657), TIPARP truncations, or point mutant constructs were co-overexpressed with pCYP1B1-luc in HuH-7 cells. Twenty-four hours after transfection, cells were treated with DMSO or 10 nM TCDD for 24 h. Corresponding western blots of transfected HuH7 cells of GFP-TIPARP proteins and βactin are shown below reporter gene data. Data were presented as means ± SEM from three independent experiments. *P < 0.05 compared with non-TIPARP-transfected cells treated with TCDD. ^#P < 0.05 compared with TIPARP-transfected cells treated with TCDD. (E) In cell mono-ADP-ribosylation of AHR by TIPARP, and different TIPARP point mutants and truncation variants. Mono-ADP-ribosylated AHR and TIPARP was detected by anti-PAN-ADPr reagent. (F) TIPARP does not mono-ADP-ribosylate ARNT in transient transfected COS1 cells. The data (E,F) are representative of three independent experiments.