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. 2016 Jul 18;126(8):3089–3103. doi: 10.1172/JCI83404

Figure 5. p21 modulates the balance between p65-p50 and p50-p50 NF-κB dimers in LPS-tolerized macrophages.

Figure 5

WT and P21–/– resting or tolerized (20 hours) peritoneal macrophages were stimulated with LPS (4 hours). NF-κB complexes that bound the consensus or the Ifnb promoter sequence were analyzed by EMSA. Anti–p50 and –p65 NF-κB antibodies were used for supershift analysis. Supershift intensity was assessed by densitometry and plotted as the percentage of supershifted complex at indicated times after LPS stimulation. (A) Left, Increased NF-κB binding to the consensus sequence in P21–/– (lane 5) compared with WT (lane 2) LPS-activated macrophages (shown at 15 minutes). Right, Relative NF-κB complex composition at different times after LPS activation. (B) Left, Similar NF-κB binding to the consensus sequence in WT (lane 2) and P21–/– (lane 5) LPS-tolerized macrophages (shown at 15 minutes) and reduced p50-p50 DNA binding in P21–/– macrophages as indicated by arrows (compare lanes 4 and 7). Right, Delayed switch to p50-p50 NF-κB complex composition in P21–/– LPS-tolerized macrophages. (C) Left, Similar NF-κB binding to the Ifnb promoter sequence in WT (lane 2) and P21–/– (lane 5) macrophages after LPS restimulation (shown at 30 minutes) and reduced p50-p50 DNA binding in P21–/– macrophages (compare lanes 4 and 7; arrow). Right, Delayed switch to p50-p50 NF-κB complexes bound to the Ifnb promoter sequence in P21–/– LPS-tolerized macrophages. In all gels, the first lane is nuclear extract of untreated macrophages (negative control). Representative results of 2 independent experiments are shown.