Anti igg horseradish peroxidase conjugate

For protein-expression analysis, whole-cell extracts were prepared from cultured cells with or without various treatments, as previously described [35 (link)]. Twenty-five or fifty μg of extract was blotted onto a PVDF filter following SDS-polyacrylamide gel electrophoresis. Anti-H2AX, anti-γH2AX (Millipore, Temecula, CA), anti-Caspase3, anti-Cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA), anti-HIF-1α (BD Pharmingen, San Diego, CA), anti-DEC1 (Novus Biologicals, Littleton, CO), anti-DEC2 (invitrogen, Rockford, IL), and anti-β-actin (Sigma) were used as primary antibodies. A mouse or rabbit anti-IgG horseradish peroxidase conjugate (Amersham Life Science) was used as a secondary antibody. Immunocomplexes were visualized with the enhanced chemiluminescence reagent ECL Plus (Amersham Life Science). Expression levels of γH2AX or Cleaved Caspase-3 were quantified by measuring densities using ImageJ (public domain open source software; imagej.nih.gov), and at least three independent measurements were averaged. Relative expression levels of γH2AX or Cleaved Caspase-3 were calculated using expression levels of H2AX or Caspase-3 as the denominator for each time point.