JIL-1 is required for phosphorylation at enhancers and promoters upon ecdysone transcriptional activation. (A) ChIP analysis of Kc167 cells treated for 3 h with ethanol (Non) or 20-HE (20HE). Antibodies used for pull-down are indicated along the x-axis. (Ser5ph) RNAPII phosphorylated on serine 5 of CTD, (Ser2ph) RNAPII phosphorylated on serine 2 of CTD. DNA was quantified by real-time PCR using primers designed to amplify the promoter, enhancer, and coding regions of each of the three genes. (B) ChIP analysis in Kc167 cells in which JIL-1 or Gal expression was inhibited by RNAi. Cells were treated with 20-HE for 3 h. Antibodies used are indicated along the x-axis. (Ser5ph) RNAPII phosphorylated on serine 5 of CTD, (Ser2ph) RNAPII phosphorylated on serine 2 of CTD. DNA was quantified by real-time PCR, and results are reported as percentage of input. (C) RNA expression analysis in cells treated with dsRNAs corresponding to the JIL-1 or Gal genes. Cells were treated with ethanol (Non) or 20-HE (20HE) for 3 h. RNA levels were determined by qPCR using primers specific to each of the four Eip75B transcripts. All samples were normalized to the mitochondrial gene myt:Col and the Non sample was set to 1 for comparison. (D) Western analysis of dsRNA-treated cells showing undetectable levels of JIL-1. Error bars represent the standard deviation of the mean of 3 biological replicates. (*) P < 0.05, (**) P < 0.01.
