Interactions between p65 and Rel-B exist in pre-labour, human amnion.

(A) Whole cell lysate from unstimulated and IL-1β stimulated pre-labour amnion epithelial cells was incubated p65 conjugated beads. The lysate was recaptured under denaturing conditions and Western immunoblotting performed with either anti-p65, anti-phospho-p65 or anti-Rel-B antibodies. Non-stimulated amnion was shown to contain both p65-pp65 and to a greater extent, p65-Rel-B dimers. When stimulated with IL-1β, p65-pp65 dimer levels increased maximally at 30 min and then decreased gradually over 24 h. Dimers of p65-Rel-B were maintained at high levels throughout the time series. (B) Non-radioactive DNA binding assay kit (TRANSAM perbioscience) measuring the binding of Rel-B to the NFκB consensus binding sequence in response to IL-1β showed an increase from the unstimulated state at 30 min before dropping slightly 1 h. Binding peaked at 4 h before again subsiding at 24 h.