Figure 12

AtRCD1 does not bind NAD and does not have ADP-ribosylation activity. Biochemical analysis of NAD binding and ART activity of AtRCD1. (A) NAD binding analysis: Autoradiography image of a SDS-PAGE gel showing proteins labeled with [³²P-NAD] upon UV irradiation. SAD-A, RCD1-GST, PCT-GST or GST were incubated with 0.6 μ M of [³²P-NAD] in absence or presence of 0.6 mM of unlabeled NAD under UV light (see Methods). (B) Picture of the SDS-PAGE gel shown in (A) stained with Coomassie Brilliant Blue. Positions of RCD1-GST and PCT-GST are marked on panels (A) and (B) with arrows; asterisks mark the position of the DnaK protein. (C) ART activity analysis: Autoradiography image of SDS-PAGE gel showing poly-ADP-ribosylation of proteins in presence of [³²P-NAD]. HsPARP1, RCD1-GST or PCT-GST in concentration 200 nM were incubated with 1.3 μ M [³²P-NAD] (see Methods) in absence or presence of 3 μ g of histones. (D) Picture of the SDS-PAGE gel shown in (C) stained with Coomassie Brilliant Blue. The 70 kDa band represents BSA used as a carrier for protein precipitation. All panels: Unlabeled NAD was used in the competition experiments. Molecular weight marker sizes (kDa) are indicated on the left side of each panel. The experiment was repeated three times with similar results, one representative experiment is shown.