FIG. 3.

Cells stably expressing A224L have elevated NF-κB activity. (A) Stimulation of NF-κB-directed transcription. Jurkat cells stably expressing A224L (shaded bars) or pcDNA as a control (open bars) were cotransfected with NF-κB (left panel) or AP-1 (right panel) luciferase reporter constructs. After 12 h of growth, the indicated cultures were exposed to PMA plus ionophore (Io) or left unstimulated. Whole-cell extracts were prepared at 24 h posttransfection and assayed for luciferase activity. Relative light units (RLU) per 0.1 μg of protein from triplicate transfections (mean ± SE) are shown. (B) A224L expression enhances NF-κB activity in the nucleus of stimulated cells. Supershift electrophoretic mobility shift assays for NF-κB DNA-binding activity were performed with nuclear extracts collected from cells not expressing or expressing A224L stimulated with PMA plus ionophore. The observed shift in mobility with specific anti-p65 antibody is indicated by an arrowhead. (C) Whole-cell extracts (50 μg) from Jurkat cells stably transfected with pcDNA or pcDNA-A224L were prepared, subjected to SDS-PAGE, and detected by immunoblotting with c-Rel-specific antibody. A representative experiment from three different assays is shown. A control using an antiactin antibody is also included to rule out differences in loading or transference. The increased amount of c-Rel in A224L-expressing cells was monitored by densitometry and is shown as optical density (O.D.).