Ku70 n3h10

Nuclear Extracts were prepared as described previously73 (link). For co-immunopreciptation experiments, extracts were adjusted to 0.15% NP-40 and 100 mM KCl, and incubated at 4 °C with either Ku70 antibody (N3H10, Santa Cruz, Santa Cruz, CA), or Ku80 antibody (C-20, Santa Cruz). Immunoprecipitates were isolated with Pierce Protein G magnetic beads (ThermoFisher Scientific, Rockford, IL). For Western blot analysis, extracts were resolved by SDS-PAGE, transferred onto a PVDF (Polyvinylidene fluoride) membrane and hybridized with the following antibodies: β-actin (I-19, Santa Cruz), Ku70 (N3H10, Santa Cruz), Ku80 (C-20, Santa Cruz), HA (HA-7, Sigma), p21 (C-19, Santa Cruz), phospho-serine 1981 ATM (Pierce, ThermoFisher Scientific), ATM (Pierce, ThermoFisher Scientific), phospho-serine 10 Histone H3 (Cell Signaling, Danvers, MA), Aurora B (H-75, Santa Cruz), FLAG (Sigma-Aldrich). The blots were developed using the Clarity Western ECL substrate (Bio-rad, Hercules, CA) and imaged on the Molecular Imager^® ChemiDocTM XRS system (Bio-Rad). Quantifications were performed using Image Lab software (Bio-Rad).

The proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane and then the blot was probed for different antibodies specific for different proteins. The following antibodies were used for western blot analyses: Ku70 (N3H10) from Santa Cruz; Ku80 (no. 2753), Bax (no. 2772S), anti-Bax antibody (6A7) (ab5714), cytochrome c oxidase subunit IV (COX IV) (no. 4844), and acetylated lysine (Ac-K-103) from Cell Signaling; and GAPDH (6C5) from Millipore and β-tubulin from Upstate. The presence of protein was visualized by using Lumigen enhanced chemiluminescence (ECL) plus PS-3.