Immunoblotting analysis of cellular proteins

Whole cell lysates obtained as previously described [13 (link)] and nuclear and cytoplasmic lysates obtained using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fischer Scientific, Waltham, MA, USA) were immunoblotted as previously described [13 (link)]. Primary antibodies to PARP1 and c-Myc (Cell Signaling Technology, Danvers, MA, USA), XRCC1 and DNA polymerase θ (Abcam, Cambridge, United Kingdom), DNA ligase 3 (BD Biosciences, San Jose, CA, USA), γ-H2AX (S139) (Millipore Sigma, Burlington, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Calbiochem), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) and vinculin (Sigma-Aldrich) were used. Antibodies to the nuclear protein histone 3 (H3) (Abcam) and the cytoplasmic protein growth factor receptor-bound protein 2 (GRB2) (BD Biosciences) were used as controls in analyses of nuclear and whole cell protein levels. Densitometry was performed with VisionWorks LS Image Acquisition and Analysis Software (UVP, Upland, CA, USA). Protein levels were normalized to β-actin. Triplicate experiments were performed.