Nub binds to the upstream region of several antimicrobial peptide genes. (A) Promoter regions of CecA1, CecC, Dipt, AttC and Act5C, in which Oct and Oct-like-binding site(s) are represented by black boxes. The numbers below each box represents the position of the binding site in correlation to the transcriptional start site. The arrows indicate the forward and reverse primers. In the Act5C gene, two Oct-binding sites are located in the first intron, while the primers bind to a region in the second intron. (B) Chromatin was extracted from whole OrR flies after fixation with formaldehyde and immunoprecipitation reactions were carried out with a peptide-specific Nub antibody (lane 4). Negative control immunoprecipitations were carried out in parallel, either without antibody (lane 2) or with an antibody against the C-terminal domain of RNA polymerase II (lane 3), which is not expected to bind to the upstream region of these genes in uninfected flies. Primers located in the transcribed region of Act5C gene and in a non-transcribed region (intergenic region) were analyzed in parallel to assess the specificity of the chromatin immunoprecipitation, showing that RNA polymerase II, but not Nub, binds to the transcribed region of the Act5C gene, while neither protein binds to the non-transcribed region. (C) Control immunoprecipitation with chromatin, which was phenol-chloroform-extracted prior to fixation, showing the specificity of the protein-antibody interactions. As expected, no PCR products were observed with any of the antibodies.
Dantoft et al. BMC Biology 2013 11:99 doi:10.1186/1741-7007-11-99