Westernsure chemiluminescent western blot reagent

Anti-XRCC4 rabbit polyclonal antibody [47 (link)], anti-FLAG mouse monoclonal antibody (clone M2; F3165; Sigma-Aldrich; St. Louis, MO, USA), anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mouse monoclonal antibody (clone 6C5; MAB374;) and anti-LIG4 guinea pig polyclonal antibody (gifted by Prof. Miki Shinohara, Kinki University) [36 (link)] were used as the primary antibody at 1/1000 to 1/5000 dilution. As the secondary antibody, horseradish peroxidase-conjugated anti-rabbit immunoglobulins swine polyclonal antibody (P0399; Dako; Glostrup, Denmark), anti-mouse immunoglobulins goat polyclonal antibody (P0447; Dako) or anti-guinea pig immunoglobulins rabbit polyclonal antibody (P0141; Dako) was used at 1/1000 to 1/3000 dilution. The immunocomplexes were developed using WesternSure Chemiluminescent Western Blot Reagent (LI-COR; Lincoln, NE, USA) and the images were captured by C-Digit Blot Scanner (LI-COR). Protein Ladder One Plus, Triple-color (Nacalai Tesque) was used as the molecular weight standard. Other procedures of western blotting followed earlier publications [31 , 32 (link)].

Western blotting was performed as described previously [32 (link)]. Anti-XRCC4 rabbit polyclonal antibody, α-XRCC4, and XRCC4 Ser320 phosphorylation-specific antibody, α-XRCC4-pS320, were generated previously [32 (link)]. Anti-PCNA rabbit polyclonal antibody (FL261) was purchased from Santa Cruz Biotechnology. Anti-rabbit immunoglobulins swine polyclonal antibody conjugated with horseradish peroxidase (P0399) was purchased from DAKO. The immunocomplexes were developed using Western Lightning ECL-Pro reagent (PerkinElmer) or WesternSure Chemiluminescent Western Blot reagent (LI-COR) and the images were captured using C-Digit Blot Scanner (LI-COR). Protein Ladder One Triple-color Broad Range (Nacalai Tesque) was used as the standard molecular mass marker.