Figure 8

Chromatin immunoprecipitation enriches for testicular HIF-1 /chromatin complexes bound to the Mcl-1 promoter. (A) Representative example of a ChIP control experiment to determine the optimum shearing time and quality of shearing over time via amplification of β-actin genomic DNA sequences in input DNA from normoxic rat testis. As a negative control, DNA was immunoprecipitated with a β-actin polyclonal antibody for the purpose of determining the specificity of ChIP assays (because β-actin is not a classic DNA-binding protein) and then subjected to PCR. (B) ChIP assays to detect HIF-1 binding in normoxic testis demonstrated complexes enriched for Mcl-1 promoter sequences when precipitated using a HIF-1α antibody (+) compared to input DNA that was not immunoprecipitated with the HIF-1α antibody (−). Negative controls in which a nonspecific IgG antibody was used for immunoprecipitation revealed no amplification of Mcl-1 promoter sequences.