Figure 4. ARTD15 is an active ADP-ribosyl transferase.

(A & C) Two µg of recombinant, purified GST-ARTD15 or GST-ARTD15-H152A/Y254A (ARTD15 dm) were [³²P]-ADP-ribosylated in vitro in the presence of increasing concentration of NAD (as indicated in A) or with 10 µM NAD (C) and analysed by autoradiography (AR). Ponceau red staining is a control of protein loading. (B) Recombinant, purified GST-ARTD15 (300 ng) was [³²P]-ADP-ribosylated and analysed by autoradiography (AR). The graph indicates the picomoles of [³²P]-ADP-ribosylated ARTD15. The inset shows the Lineweaver-Burk analysis of the data. The data shown are the means (±SD) of four independent experiments performed in triplicate. (D) Total membrane proteins (50 µg) obtained from HeLa cells transiently transfected with empty vector (control), ARTD15 or ARTC1, as indicated, were [³²P]-ADP-ribosylated in vitro in the absence (left) or presence (right) of 1 mM agmatine. The supernatants and, as a further control, [³²P]-NAD (NAD) and [³²P]-ADP-ribose (NADase) were analysed by TLC and autoradiography. The data shown are representative of at least five independent experiments. (E) Two µg of recombinant, purified GST-ARTD15 (ARTD15) or plasma membranes added with 250 ng of purified ßγ dimer (ß subunit) were ADP-ribosylated with [³²P]-NAD⁺ for 1 h at 37°C, in the presence or absence of MIBG. The loading control is shown (WB). Data shown are representative of at least three independent experiments. (F) ADP-ribosylated ARTD15 was blotted and the filters were treated with the indicated compounds. Data reported in the graph are the mean (±SD) of three independent experiments performed in duplicate (control: untreated sample). The inset shows a representative experiment.