Figure 3

Treatment of HeLa cells with cGMP agonist, 8-(4-chlorophenylthio)guanosine 3'.5'-cyclic monophosphate (8-pCPT-cGMP), increased NF-κβ p65 in the nucleus. A) HeLa cells were mock treated or treated with TNFa or 8-pCPT-cGMP as indicated. Nuclear and cytoplasmic fractions were collected and assayed for NF-κβ p65 by Western blotting. Both TNFα and 8-pCPT-cGMP were found to increase nuclear NF-κβ p65 while commensurately decreasing cytoplasmic NF-κβ p65. Western blotting for tubulin controlled for subcellular fractionations. Quantifications of relative distributions of NF-κβ p65 are shown at bottom. B) PKG1β increases expression of NF-κB dependent luciferase. HeLa cells were transfected with NF-κB luciferase reporter plasmid in the presence or absence of co-transfected PKG1β expression plasmid. Cells were harvested 24 hours post-transfection, and cell lysates were assayed for luciferase activity as indicated.