Extended Data Figure 8: A20 OTU domain, but not the ZnF4 motif, downmodulates LPS signalling.

a, Kaplan–Meier survival curves of A20 WT (n = 15) and A20 OTU(C103A) (n = 15) mice in response to 20 mg LPS per kg body weight. Log rank P = 0.0002, Wilcoxon P < 0.0001. b, Upper panel, analysis of TRAF6 K63 ubiquitination (Ub) status in LPS-treated WT and A20 OTU(C103A) primary BMDMs. Lower panels, immunoblot analysis of whole-cell lysates from LPS-treated wild-type and A20 OTU(C103A) primary BMDMs. Asterisk, background band. Similar trends were seen in wild-type and A20 OTU(C103A) MEFs in response to acute LPS treatment and following LPS pre-treatment to induce A20 expression (not shown). c, Kaplan–Meier survival curves of A20 wild-type (n = 10), A20 ZnF4(C609A,C612A) (n = 10), and A20 ZnF4(Y599A,F600A) (n = 9) mice in response to 20 mg LPS per kg body weight. Log rank P = 0.1531, Wilcoxon P = 0.1398 for A20 ZnF4(C609A,C612A) versus WT; Log rank P = 0.4103, Wilcoxon P = 0.3373 for A20 ZnF4(Y599A,F600A) versus WT. d, A heat map representing profiles of serum cytokines in 12 different genotype/LPS treatment groups. Mice (n = 3 or 4 per group) were treated for the indicated time with 40 mg LPS per kg body weight LPS as indicated; mean values per group are represented in the heat map. Each row represents one cytokine, whose values were standardized to z-scores with a mean of zero and a standard deviation of 1, and colour-coded according to the colour key. Variances of selected serum cytokines from A20 WT, A20 OTU(C103A) (OTU), or A20 ZnF4(C609A,C612A) (ZnF4 Cys) mice in response to LPS stimulation were evaluated using the Student’s t-test: TNF WT versus OTU 2 h P = 0.044; IFNγ WT versus OTU 4 h P = 0.033; Ccl4 WT versus OTU 4 h P = 0.040. Profiles of selected serum cytokines from A20 WT (n = 5), ZnF4 Cys (n = 4), or OTU(C103A) (n = 5) mice in response to PBS or low dose (5 mg LPS per kg body weight) LPS and collected at 2 h or 6 h post-stimulation showed similarly significant variances between A20 WT and A20 OTU(C103A) (OTU) but not between A20 WT and A20 ZnF4(C609A,C612A) (ZnF4 Cys) (not shown). e, Upper panel, immunoblot analysis of whole-cell lysates from LPS-treated wild-type A20 and A20 ZnF4 C609,612A MEFs. Lower panel, analysis of TRAF6 K63 ubiquitination (Ub) status in the corresponding MEFs. Similar trends were seen in lysates from LPS-treated wild-type and A20 ZnF4 C609,612A MEFs following LPS pre-treatment to induce A20 expression (not shown). For gel source data, see Supplementary Fig. 10. Data represent two biological replicates.