Synergistic Activations of REG Iα and REG Iβ Promoters by IL-6 and Glucocorticoids through JAK/STAT Pathway in Human Pancreatic β Cells

Figure 5

IL-6 + Dx-increased STAT3 binding to the REG Iα promoter. (a) Detection of DNA-protein complexes using EMSA. Nuclear extracts (2.5 μg protein) prepared from 1.1B4 cells, treated without or with Dx (100 nM), or IL-6 (20 ng/mL), or IL-6 + Dx, were incubated with a ³²P-labeled probe 1. Nuclear extracts from untreated cells were applied onto lanes 1, 5, and 9; those from Dx-treated cells were applied onto lanes 2, 6, and 10; those from IL-6-treated cells were applied onto lanes 3, 7, and 11; and those from IL-6 + Dx-treated cells were applied onto lanes 4, 8, and 12. Lanes 1–4 contain no competitor; lanes 5–8 contain 100 × unlabeled probe 1; lanes 9–12 contain 100 × unlabeled probe M1. An arrow marks IL-6 + Dx-inducible complex migration. (b) Supershift assay of STAT3 containing complex. EMSAs were performed in the presence or absence of STAT3 antibodies as indicated. An arrow marks IL-6 + Dx-inducible complex migration. An arrowhead indicates supershift complex by anti-STAT3 antibody. (c) ChIP assay showing IL-6 + Dx increases in STAT3 binding to the REG Iα promoter. 1.1B4 cells were treated without or with IL-6 (20 ng/mL) + Dx (100 nM). ChIPs were performed with anti-STAT3 antibody, followed by PCR with primers specific for the REG Iα promoter. A representative result from three experiments is shown.

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