14-3-3 recruitment and H3K27 acetylation are dependent on JIL-1 kinase at enhancers and promoters. (A) Kc167 cells treated with dsRNAs to Gal or JIL-1 were incubated with 20-HE for 3 h and subjected to ChIP analysis using the antibodies indicated along the x-axis. DNA was quantified by real-time PCR and reported as a percent of the input. (B) Kc167 cell lysates with and without phosphatase inhibitors (PPI) were immunoprecipitated using antibodies against JIL-1, loaded 1:10 with respect to input, and subjected to Western analysis using antibodies against JIL-1, CBP, and 14-3-3. (C) Using Kc167 cells pretreated for 5 min with 100 μM DRB followed by 3-h treatment with 20-HE (20HE), ChIP was performed using the antibodies indicated along the x-axis and quantitated by real-time PCR using primers designed to amplify the promoter, enhancer, and coding regions of the Eip75B gene. (D) Relative expression analysis of the DRB-treated cells compared with normal 20-HE induction using primers specific to each of the four transcripts of Eip75B. All samples were normalized to mitochondrial gene product myt:Col and the Non sample set to 1 for comparison. Error bars represent the standard deviation of the mean of 3 biological replicates. (E) Western analysis of lysates from Kc167 cells treated with dsRNAs corresponding to the JIL-1, CBP, 14-3-3, or Gal genes; cells were pretreated with DRB for 5 min and with 20-HE for 3 h. Error bars represent the standard deviation of the mean of 3 biological replicates. (*) P < 0.05, (**) P < 0.01.
