Figure 6.

Regulation of STAT-1 DNA binding activity by lipopolysaccharide (LPS), tumour necrosis factor-α (TNF-α) and retinoic acid (RA). Cells were pretreated with RA for 16 hr and then treated with LPS or/and TNF-α for 2 or 24 hr in the presence and absence of fresh RA. Nuclear protein was isolated and tested for binding activity with an ISRE/GAS DNA element. (a) EMSA showing the protein–DNA binding complex formation after cell stimulation. (b) Western blot of STAT-1 PY⁷⁰¹ from nuclear proteins extracted from 24-hr-treated samples. (c) Competition assay and supershift assay showing the specificity of the binding complex. Nuclear protein extract used was from the 24 hr LPS plus TNF-α treated sample. Abbreviations used in figure: NPE, nuclear protein extract; W, wild-type; Mu, mutant type; NS, non-specific complex. Sp-1 was used as a non-related DNA competitor, and anti-iNOS (inducible nitric oxide synthase) antibody was used as a non-related antibody for supershift assay.