Figure 3.

Rel members are capable of mediating transcriptional activation through the Bcl-x promoter by binding to NF-κB cis elements. (A) The NF-κB complex binding to the putative NF-κB-binding sites within the Bcl-x promoter consists predominantly of p50, p65, and c-Rel. Nuclear extract harvests and binding reactions were performed as described previously. Supershifts were performed by the addition of the appropriate antibody to the nuclear extracts on ice 20 min before the binding reaction. (B) p65 is capable of mediating transcriptional activation through the Bcl-x promoter. A Bcl-x promoter fragment spanning −640 to +9 relative to the transcriptional start site (Bcl-x WT promoter) and another fragment missing the NF-κB-binding sequence (Bcl-x κB- promoter) were cloned into the pGL2-Basic luciferase reporter vector. 293T cells were transfected with 100 ng of the indicated reporter plasmids and either 2 μg of p65/RelA expression vector or 2 μg of pBABE-puro control vector as carrier. Samples were harvested 36 hr after transfection and assessed for luciferase activity.