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

Figure 6. p21 regulates p50-p50 NF-κB DNA binding.

Figure 6

(A) RT-PCR analysis showing similar Nfkb1 (encoding p105) and Bcl3 gene expression in WT and P21–/– LPS-tolerized/restimulated peritoneal macrophages. Results were normalized to β‑actin and show fold induction over unstimulated WT cells. Data are representative of 3 independent experiments. (B) Immunoblot showing similar p50 protein levels in the cytoplasmic and nuclear fractions of WT and P21–/– LPS-tolerized/restimulated peritoneal macrophages. β-Actin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. (C) Reduced ubiquitination of p50 in P21–/– LPS-tolerized/restimulated peritoneal macrophages. Equal amounts of protein were immunoprecipitated with antibody against p50 and immunoblotted with antibody against ubiquitin. n.c., negative control; IP performed in the absence of cell extracts. (D) Reduced p50 phosphorylation in P21–/– LPS-tolerized/restimulated peritoneal macrophages. Equal amounts of protein were immunoprecipitated with antibody against p50 and immunoblotted with antibody against phospho-serine/threonine. (E) p21 increases p50 homodimer binding to DNA by increasing its affinity. Tolerized (20 hours) WT and P21–/– peritoneal macrophages were stimulated with LPS (15 minutes). Anti–p50 NF-κB antibody was used for supershift assays with increasing amounts of unlabeled oligonucleotide (cold probe), and NF-κB complexes binding the Ifnb promoter sequence were analyzed by EMSA. (F) p50-p50 and p65-p50 DNA binding was measured by densitometry of autoradiogram in E. Shown are representative gels of at least 2 experiments performed.