Figure 5

The mR3-3 c-Myb mutant has impaired chromatin binding. A. Western analysis was used to determine the amounts of core histones present in the vector only control (v/o), wild-type c-Myb (wt), and mR3-3 (mR3) chromatin immunoprecipitation fractions. Data displayed used antibodies directed at histone H3 (Abcam) and histone H4 (Upstate). Input and chromatin immunoprecipitation (ChIP) fractions are shown at different exposures. B. The relative amounts of core histones from several blots similar to those shown in (A) were quantitated and normalized for background using the ImageJ program http://rsb.info.nih.gov/ij/. Wild-type levels were set to 100% and the mutants were normalized to this level for adequate comparison between different experiments. The plots shown represent an average of two separate experiments for H2A and five separate experiments for H3. The H4 antibody was used for only one experiment but is consistent with results for the other core histones. C. We compared the amount of H3-acetyl R18 (H3-AcR18) and H3-acetyl K23 (H3-AcK23) modifications present in the wild-type and mR3-3 bound nucleosomes using specific anti-H3-acetyl R18 and K23 antibodies (Upstate). Labeled as in A. Inputs are shown at a different exposure than chromatin immunoprecipitation (ChIP) samples and confirm that samples have similar amounts of input protein.