Figure 4.

Role of ATF4 in PCAF recruitment to CHOP AARE in response to leucine starvation. ATF4 +/+ and ATF4 −/− MEF were incubated 2 h either in control (+leu) or leucine-free medium (−leu) and harvested. (A) ChIP analysis was performed as described under Materials and Methods section using antibodies specific for PCAF and ATF4 and a set of primers to produce amplicon B (Figure 3A). Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments and the error bars represent the SEM. We note that there remains 5% of ATF4 antibody binding in ATF4 KO cells. ChIP experiments were also performed with primer sets reaching much farther upstream or downstream from the CHOP gene (data not shown). The results indicate that the amount of ATF4 binding in ATF4-deficient cells was due to the background observed for the ATF4 antibody. (B) Western blot analysis of ATF4 and PCAF was performed from nuclear extracts.