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. 2000 Jul 24;150(2):293–308. doi: 10.1083/jcb.150.2.293

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Electrical activity inducing PARP activation in brain cortical neurons. (a) Evoked action potentials and synaptic potentials, recorded by the patch-clamp whole cell technique in the current clamp mode in cultured cortical neurons during electrical stimulation (n = 4). (Top) stimulation inducing polyADP-ribosylation of nuclear proteins (see b): 2-s repetitive (100 Hz) 30-volt, 0.1 ms pulses. Evoked action potentials, underlined by a bar, are presented on an expanded time-base to the right. The break in the trace represents a gap of 12 s. (Bottom) A stimulation which did not induce polyADP-ribosylation: 2-s repetitive (1 Hz) 30-volt, 0.1 ms pulses. (b) (Top) Autoradiograms of [³²P]polyADP-ribosylated PARP in crude nuclei isolated from cortical neurons pretreated as follows: lane 1, unstimulated neurons; lanes 3, 5, 7, and 9, neurons repolarized for 20 min after the following depolarizations (respectively): high-[K⁺] for 5 min (lane 2), a 10-min train of repetitive (10 Hz) 30-volt, 0.1 ms pulses (lane 4), a 2-min train of repetitive (100 Hz) 30-volt, 0.1 ms pulses (lane 6), and repetitive (100 Hz) 30-volt, 0.1 ms pulses, applied for 2 s every minute for 10 min (lane 8). PARP was immunoprecipitated from the nuclear protein extracts by N-20 antibody, subjected to SDS-PAGE, autoradiographed, and electroblotted (Western blot). (Bottom) Immunolabeling of [³²P]polyADP-ribosylated PARP by anti-PARP Vic-5 antibody in the immunoprecipitates (n = 8).