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. 2021 Apr 19;49(9):5003–5016. doi: 10.1093/nar/gkab269

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — LIG1 deficiency causes histone H3 ADP-ribosylation. (A) Mock- and xLIG1-depleted extracts were used for replication of sperm chromatin. xLIG1-depleted extracts were supplemented with wild-type xLIG1 or xLIG1-K721A. Chromatin-bound proteins were analyzed by immunoblotting using the indicated antibodies and pan ADP-ribose (Pan-ADPr) detecting reagent (n = 3). The asterisk indicates a non-specific band. (B) Mock-, xLIG1-, xPARG- and xLIG1/xPARG-depleted extracts were used for replication of sperm nuclei. Chromatin-bound proteins were analyzed as in Figure 3A (n = 2). (C) Chromatin fractions from the indicated immunodepleted extracts were solubilized with MNase followed by immunoprecipitation with anti-histone H3 antibodies after treatment with 1% SDS. The resultant immunoprecipitates were analyzed as in Figure 3A (n = 2). (D) Mock- and xLIG1-depleted extracts were used for replication of sperm nuclei in the presence of 30 μM APH where indicated. Chromatin-bound proteins were analyzed by immunoblotting using the indicated antibodies and pan ADP-ribose detecting reagent (n = 2). (E) Mock- and xLIG1-depleted extracts were used for replication of sperm chromatin. xLIG1-depleted extracts were supplemented with either buffer or recombinant wild-type His10-xARH3 or His10-xARH3-D58N, (0.2–1 μM). Chromatin-bound proteins were analyzed by immunoblotting using the indicated antibodies and pan ADP-ribose detecting reagent (n = 2).