Fig. 4.
NMNAT-1 associates with PAR and automodified PARP-1 and accelerates poly(ADP-ribosyl)ation and AIF translocation. (A) Specific interaction of NMNAT-1 with free or PARP-1-bound PAR was visualized by spotting the indicated proteins (0.5 μg) on a nitrocellulose membrane. The membrane was then incubated with 32P-labeled oligo(ADP-ribosyl)ated PARP-1 (oPARP-1, 0.25 μg), poly(ADP-ribosyl)ated PARP-1 (pPARP-1, 0.25 μg), or protein-free PAR (PAR, 5 μM). After washing, specific binding was visualized by autoradiography. (B) PARP-1 (25 ng) was incubated with 32P-NAD+ in the absence or presence of NMNAT-1 in a mass ratio (PARP-1/NMNAT-1) of 1:2, 1:4, 1:10, or 1:20. Thereafter, the proteins were separated by SDS/PAGE. (Top) The autoradiograph of the gel is shown. (Middle and Bottom) Proteins were visualized by the fluorescent SYPRO Ruby stain (Invitrogen, Carlsbad, CA). (C) PARP-1 (100 ng) was incubated with nicotinamide/[14C]NAD+ in the presence or absence of NMNAT-1 (mass ratio of 1:5). [14C]Nicotinamide released during automodification of PARP-1 was identified by HPLC using radioisotope detection. (D) PAR-mediated AIF translocation is enhanced by NMNAT-1 overexpression. FLAG-tagged NMNAT-1 was transiently overexpressed in HeLa cells. (Bottom Left) As a control, nuclear EGFP was overexpressed. The cells were treated with 100 μM H2O2 as indicated. FLAG-tagged NMNAT-1 and AIF were visualized by immunostaining.