Anti xlf

Whole cell extracts were obtained using NETN lysis buffer. The proteins were separated by 10% SDS-PAGE and detected using the following primary antibodies according to the manufacturer's instructions: anti-XRCC4 (Abcam, MA), anti-XLF (Abcam, MA), anti-Ligase IV (Thermo Scientific), anti-FLAG (M2, Sigma-Aldrich), anti-GFP (Invitrogen), and anti-GAPDH (Proteintech).

The following antibodies were used for western blotting or immunoprecipitation: anti-XRCC4 (mouse, 611506, BD), anti-CtIP (sc5970, Santa Cruz Biotechnology), anti-XRCC3 (sc53471, Santa Cruz Biotechnology), anti-XLF (ab33499, Abcam), anti-tubulin (sc5286, Santa Cruz Biotechnology), anti-phospho-histone H3 (S10) (06-570, Millipore), anti-FLAG (1E6, Wako), anti-DNA ligase IV (this study) and anti-pS326 of XRCC4 (this study). Antibodies used for immunostaining were as follows: anti-53BP1 (NB100-304, Novus Biologicals), anti-Rad51 [61] (link), anti-XRCC4 (this study), and anti-DNA ligase IV (this study). For preparation of antibodies against XRCC4 and DNA ligase IV, full-length human XRCC4 tagged with hexahistidine and a 367-residue peptide containing the C-terminus of human DNA ligase IV tagged with hexahistidine were affinity purified from Escherichia coli with a nickel/cobalt column and used for immunization of rat or guinea pig, respectively. Anti-pS326 was raised in rabbits against a synthesized phosphopeptide, TLRNSpSPEDLFC. Post-immune IgG was affinity purified with this phosphopeptide and also titrated using a non-phosphorylated peptide, TLRNSSPEDLFC (custom-made by MBL Co., Ltd.). Immunization and preparation of antisera were carried out by MBL Co., Ltd.

Paraffin breast tumor sections were subjected to immunohistochemical staining to ascertain the protein localization of XRCC4, XLF, and Ligase IV. The tumor sections were incubated with the following primary antibodies: anti-XRCC4 (1:500; Abcam, MA), anti-XLF (1:400; Abcam, MA), and anti-Ligase IV (1:600; Thermo Scientific). HRP-conjugated secondary antibodies were used, followed by colorimetric detection using an Envision detection kit (DAKO, Japan).