Rab27b may be a direct transcriptional target of NF-E2. (A) Four potential NF-E2 binding sites, here designated A-D, are identified between 1 kb upstream of the transcription start site (designated position +1) and the end of the first intron in the mouse Rab27b gene locus, using software that detects consensus matches in nucleotide sequence (http://www.genomatix.de/matinspector). (B) Chromatin immunoprecipitation (ChIP) analysis of primary MKs using p45 NF-E2 antibody indicates that NF-E2 is recruited to the A site of the Rab27b locus. PCR products were detected on ethidium bromide-stained agarose gels. Immunoprecipitating antibodies and conditions are indicated as follows: no chr indicates no chromatin; no Ab, none; p45, rabbit p45 NF-E2 antiserum; and PI, preimmune serum. The adjacent panels show an additional control with absence or presence of excess peptide antigen as a competitor in the immunoprecipitation and a histogram of quantitation of the visible bands in units of relative pixel density. The results shown are representative of 5 independent experiments. (C) Sequence and location of 4 potential NF-E2 binding sites, designated A-D, identified between 1 kb upstream of the transcription start site (designated +1) and the end of the first intron in the mouse Rab8 gene. (D) ChIP analysis of primary MKs using a different p45 NF-E2 antibody verifies that NF-E2 is recruited to the A site in the Rab27b promoter (quantified in the right panel) but not 2 kb upstream (Rab27bNFE2UP) or downstream (Rab27bNFE2DNS) in the locus or to potential sites in the Rab8 gene.