Fig. 5

Binding of PU.1 to the hDectin-1 gene promoter. a Predicted PU.1 transcription factor-binding sites (TFBSs) within the proximal 2000 bp of the hDectin-1 gene promoter were identified. Potential binding sites were analyzed, including A (GGGAAGT, –1053 to –1047), B (AGGCAGT, –719 to –713), C (AGAAAAT, –527 to –521), D (AGAAAGA, –1411 to –1405), and E (AGAAAGA, –618 to –612). b Chromatin immunoprecipitation (ChIP) assays of PU.1-binding sites. Chromatins from THP-1 cells were prepared. Shown are the PCR products from: Lane 1, Chromatin incubated with rabbit anti-PU.1 antibodies (H-135); Lane 2, Chromatin incubated with positive control (anti-RNA Polymerase antibody); Lane 3, Chromatin incubated with negative control (normal rabbit IgG); Lane 4, Chromatin before immunoprecipitation (input chromatin). (c, d) Electrophoretic mobility shift assays (EMSA). Nuclear extracts from THP-1 cells cultured at control conditions or in the presence of PMA were subjected to EMSA. The use of biotin-labeled oligonucleotide probes A, B, and C, carrying the hDectin-1 gene promoter regions A (–1053 to –1047), B (–719 to –713), and C (–527 to –521), respectively, allowed to assess the specificity of the protein-DNA binding. The CD11b probe, which corresponded to the –22 to –12 bp PU.1 consensus sequence located on the CD11b promoter, was the positive control. Competitors were unlabeled oligonucleotide probes, and their concentrations were 25-fold and 100-fold. The NF-kB probe was the input probe, and the unrelated were nuclear extracts with active NF-kB protein. Specific binding: protein-DNA complexes of PU.1 or NF-kB proteins and the probes. NSB: nonspecific binding. Free probes: free biotin-labeled oligonucleotide probes that did not bind to the proteins. The experiments were repeated at least thrice, and the data from one representative experiment with two technical repeats are presented (*, p < 0.05)