Regulation of Il8rb transcription by G-CSF–responsive STAT3 signaling. (A) A schematic of the Il8rb gene showing the location of the STATx site (top) and sequence of the mutant STAT element (bottom). (B) 32D.G-CSFR cells were electroporated with WT (WT STAT) or mutant pGL3-Il8rb (mutant STAT) and pTK-Renilla reporter plasmids, treated with or without G-CSF for 6 hours, and assayed for luciferase activity. The ratio of firefly:renilla relative light units (RLU) in G-CSF–treated cells relative to unstimulated cells (−G-CSF) was averaged from 3 independent experiments. (C) EMSAs were performed with nuclear extracts from 32D.G-CSFR cells, stimulated with or without G-CSF for 30 minutes by the use of radiolabeled oligonucleotide probes corresponding to the Il8rb promoter region containing the STATx site (WT Il8rb) or an oligonucleotide containing a mutation in the STATx site of the Il8rb promoter (mutant Il8rb), as indicated. An excess of unlabeled oligonucleotide, corresponding to the WT Il8rb probe (Il8rb), an oligonucleotide encompassing the STAT3 binding site in Socs3 (Socs3), or an oligonucleotide containing a mutation in the STAT3 site from Socs3 (mSocs3) was used as competitor, as indicated. Results are representative of 3 independent experiments. (D) EMSAs were performed as in panel C with the WT Il8rb probe in the absence or presence of anti-STAT3 antibody. Results are representative of 3 independent experiments. (E) ChIPs were performed on 32D.G-CSFR cells ± G-CSF treatment for 30 minutes with anti-STAT3 or Ig control antibody, as indicated. Purified DNA samples were subjected to PCR to detect Il8rb (top) or Socs3 (bottom) promoter sequences. Data are representative of 3 independent experiments. Error bars represent SEM.