Figure 2.

KDM5A interacts with PARP1 and PAR chains. (A) Endogenous KDM5A and PARP1 interact. KDM5A was immunoprecipitated from HEK293T cells with or without 10-Gy IR. Samples were evaluated by WB analysis with the indicated antibodies. (B) Reciprocal IP with endogenous PARP1 was performed as in A. (C) KDM5A binds to PAR in cells in a PARP-dependent manner. HEK293T cells were treated with olaparib (5 µM, 1 h) followed by 10-Gy IR or hydrogen peroxide (H₂O₂; 1 mM, 15 min) and examined by IP-WB analysis with an anti-PAR antibody. (D and E) KDM5A binds PAR noncovalently. U2OS cells were treated with 10-Gy IR for 2 min, and cell lysates were incubated with KDM5A or IgG antibodies with or without 1% SDS. Purified samples were either blotted on nitrocellulose membrane to detect PAR chains or resolved on SDS-PAGE gel and immunoblotted to detect KDM5A. In E, experiments were performed as in D in cells expressing GFP, GFP-KDM5A, or GFP-PARP1. Immunoprecipitated samples with or without SDS were blotted onto nitrocellulose membrane and detected with anti-PAR and anti-GFP for loading. (F) Purified KDM5A from U2OS cells binds PAR chains in vitro. Purified proteins were spotted onto nitrocellulose membrane and probed with purified PAR chains with binding detected with an anti-PAR antibody.