Skip to main content
. 2021 Oct 30;49(20):11728–11745. doi: 10.1093/nar/gkab980

Figure 3.

Figure 3.

Phospho-Ku70 and mutant forms of Ku70 interact with core components of cNHEJ but only pKu70 recruits RNA Pol II in the repair complex. (A) Representative western blot of co-immunoprecipitation using anti-phospho-Ku70 shows that pKu70 interacts with the Ku80, ligase 4 and Paxx. (B) Similarly, using the anti-Ku70 antibody shows that all Ku70 forms (wild-type and mutants) interact with Ku80, ligase 4 and Paxx. The membranes were cut according to the corresponding molecular weights and probed with antibodies as previously described (see the Materials and Methods section). (C) Whole-cell protein extracts of transfected HME cells (46), expressing ser-Ku70 at 2 h post-irradiation (2 Gy), were co-immunoprecitpitated with anti-Ku70 or anti-pKu70 antibodies, resolved by SDS-PAGE and proteolyzed with trypsin. Peptides were analysed using a label-free-quantification (LFQ) approach with an orbitrap-based mass spectrometry analyser. The analysis was performed with Perseus software by varying FDR and S0 simultaneously. We chose values of S0 = 0.1 and FDR = 0.05, as the most commonly used values for this type of analysis. The corresponding genes, indicated by grey squares, are considered to exhibit non-significant differences in their affinity toward Ku70 and/or pKu70, whereas those in red squares are presumed to be more specific partners of total Ku70 or pKu70. (D) RNA Pol II localizes with γ-H2AX at laser microirradiation-induced DNA damage only in cells expressing phosphorylable ser-Ku70. Cells esxpressing ser-Ku70 or ala-Ku70 were irradiated by laser Chaameleon Vision II system and 1 min postirradiation cells were fixed and probed with anti-γ-H2AX (red) and anti-phospho-ser5-RNA Pol II (green). Hoechst 33342 was used to stain chromatin DNA. Scale bar = 5 μm. (E) RNA Pol II is recruited by pKu70 in the repair complex. Western blot analysis of immunopurified proteins from cells expressing ser-Ku70 or ala-Ku70. Cells were irradiated at 4Gy or left unirradiated, and following 30 min of post-irradiation culture, cells were lysed. The protein extracts were immunopurified by using a monoclonal anti-Ku70 antibody (clone N3H10) and magnetic beads coated with anti-mouse IgG (Estapor, Merck-Millipore). After SDS-PAGE, the membranes were probed with anti-RNA Pol II or anti-Ku70 antibodies.