Figure 6. RelB inhibits NF-κB activation via physical interaction with RelA.
A, BV-2 cells (8×105) were treated with Tat (100nM) for the indicated periods of time. Whole cell lysates were subjected to immunoprecipitation using a RelA-specific antibody and Protein A/G+ agarose beads. Immunocomplexes were separated by 7.5% SDS-PAGE and blotted onto nitrocellulose membrane and subjected to immunoblot analysis with antibodies specific for RelB or RelA. The plot below the bands represents densitometry values for each band, normalized as a ratio of RelA(IP)/RelB(IB) (i.e. top bands): RelA(IP)/RelA(IB) (i.e. bottom bands), to indicate the increase in RelB/RelA interactions at each time point. B, BV-2 cells (1.2×105) were treated with Tat (100nM) for the indicated periods of time and whole cell lysates were subjected to immunoblot analysis using either RelA-specific (upper panel), RelA phospho-serine 276-specific (center panel) or α-Tubulin-specific (lower panel) antibodies. Protein levels were quantified using ImageJ software (bottom graphs). The results of a single representative experiment are shown. C, BV-2 cells (5×106) were transiently transfected using Nucleofector (Amaxa/Lonza) with an NF-κB-dependent luciferase reporter plasmid either alone or together with a RelB-encoding plasmid. 16h post-transfection cells were either left untreated or were treated with Tat (100nM) for 8h. Luciferase activity in whole cell lysates was determined. Results are shown as mean ± SEM of values derived from three replicates from one representative experiment; two total experiments were performed. Statistical significance (p<0.001) is indicated (***). D, BV-2 cells (1.5×105) were transiently transfected using Lipofectamine (Invitrogen) with the NF-κB-luciferase reporter plasmid together with a plasmid for RelA in the absence or presence of increasing amounts of RelB-encoding plasmid. The luciferase activity in whole cell lysates was determined 18h post-transfection. Results are shown as mean ± SEM of values derived from two replicates from one representative experiment; two total experiments were performed.