Mouse anti ku70 monoclonal antibody e 5

Local DNA damage was induced through a microlaser, and subsequent cell imaging was performed as previously outlined^{51 ,56 (link)}. In brief, DSBs were generated locally using a 405 nm diode laser equipped with an FV300 CLSM system (Olympus). Live and fixed cells expressing EYFP-chicken Ku70, EYFP-chicken XLF, or EYFP alone were imaged using an FV300 CLSM system (Olympus). Immunocytochemistry was conducted as previously described^{51 ,55 (link),56 (link)} with the use of the following antibodies: mouse anti-Ku70 monoclonal antibody (E-5, Santa Cruz Biotechnology), mouse anti-Ku80 monoclonal antibody (B-4, Santa Cruz Biotechnology), mouse anti-Ku70/Ku80 monoclonal antibody (162, NeoMarkers, Fremont, CA, USA), mouse anti-γH2AX monoclonal antibody (JBW301, Merck Millipore, Billerica, USA), and donkey anti-mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555 (Thermo Fisher Scientific).

The preparation of total protein extracts and western blot analysis was conducted as previously described^{55 (link)–57 (link)}, with some modifications. Specifically, the total proteins (50 μg per lane) were separated on an Extra PAGE One Precast Gel 5–20% (Nacalai Tesque). Subsequently, the membranes were blocked with Blocking One (Nacalai Tesque) for 30 min at room temperature. The following antibodies were employed: mouse anti-Ku70 monoclonal antibody (E-5, Santa Cruz Biotechnology, Santa Cruz, TX, USA), mouse anti-Ku80 monoclonal antibody (B-4, Santa Cruz Biotechnology), rabbit anti-GFP polyclonal antibody (FL, Santa Cruz Biotechnology), and mouse anti-β-actin monoclonal antibody (AC-15, Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies used included anti-mouse IgG, horseradish peroxidase (HRP)-linked whole antibody from sheep (GE Healthcare Bio-Sci. Corp., Piscataway, NJ, USA) or anti-rabbit IgG, HRP-linked whole antibody from donkey (GE Healthcare Bio-Sci. Corp.). Protein bands were detected following the manufacturer's instructions using Chemi-Lumi One Ultra (Nacalai Tesque) and visualized with the ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA). The 3-Color prestained XL-ladder (APRO Science, Tokushima, Japan) served as the molecular weight marker.