Figure 6. A20’s ZF7 motif synergizes with A20’s ZF4 motif to restrict inflammation in vitro and in vivo.

(a) Scheme of targeting ZF7^FG residues in A20^ZF4 embryos. Generation of A20^ZF4ZF7 mice described in METHODS.

(b) Quantitation of live A20^ZF4ZF7 pups at 14 days of age born from intercrossed A20^ZF4ZF7/+ heterozygote parents.

(c) Weights of 14 day old A20^{ZF4ZF7/ZF4ZF7} and WT littermate mice. **** p < 0.0001. Unpaired two-tailed t-test.

(d) Quantitative PCR analyses of indicated genes in MEFs transiently stimulated with TNF for 15 min and measured at the indicated times. WT (black), ZF7 (red), ZF4ZF7 (purple) MEFs are shown. Mean ± SD for technical replicates. Results are representative from three independent experiments.

(e) IKK kinase assays of TNF-stimulated MEFs of the indicated genotypes. Lysates were immunoprecipitated with anti-IKKγ, incubated with GST-IκBα and assayed for phospho-IκBα at the indicated times after warming cells to 37 °C. GST and IKKγ levels in IPs shown as IP control. Immunoblot analyses of phospho-IKKα/β, phospho-IκBα and control signaling proteins in whole cell lysates shown in input samples.

(f) Immunoblot analyses of A20 expression in MEFs of indicated genotypes before and 60 min after TNF stimulation. Representative of 3 independent experiments.

(g, h) ELISA of IL-6 (g) and IL-1β (h) secretion from indicated genotypes of BMDMs after LPS stimulation. WT (black), ZF4 (blue), ZF7 (red), ZF4ZF7 (purple) mice. Biologically independent culture samples. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA, Tukey’s Multiple Comparison Test.