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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Nat Immunol. 2015 May 4;16(6):618–627. doi: 10.1038/ni.3172

Figure 6. A20 utilizes its C103 motif to inhibit RIPK1-RIPK3 complexes.

Figure 6

(a) A20 mediated deubiquitination of K63-ubiquitinated RIPK3. 293T cells were transfected with plasmids expressing RIPK3, HA-K63-Ub (HA-tagged ubiquitin in which all ubiquitin lysines except K63 were mutated to arginine), and FLAG-A20, immunoprecipitated with anti-RIPK3, and immunoblotted for HA (K63-ubiquitin) and A20. Immunoblots of whole cell lysates for RIPK3 and A20 are shown below as controls. (b) K63-ubiquitination of RIPK3 in A20−/− cells. A20+/+ and A20−/− MEFs were stimulated with TNF, CHX and ZVAD (TCZ) (“stim +”), and lysed under denaturing conditions (6M urea). Lysates were diluted, imunoprecipitated with anti-K63 ubiquitin antibody, and analyzed for RIPK3 expression. Short exposure of RIPK3 immunoblot shown to demonstrate increased and phosphorylated RIPK3; long exposure shown to demonstrate increased K63-ubiquitination of RIPK3. Immunoblots of whole cell lysates for RIPK3, A20 and actin are shown on whole cell lysates below as controls. Note the increased K63-ubiquitinated RIPK3 in stimulated A20−/− cells. Data are representative of 3 independent experiments. (c) Cell survival of the indicated genotypes of MEFs after stimulation with TNF, CHX and ZVAD (TCZ) or TNF, CHX, ZVAD and Nec1 (TCZN) for five hours. Bars indicate standard deviation. * indicates p < 0.05 by ANOVA when compared with WT cells. (d) RIPK1-RIPK3 complexes in indicated genotypres of MEFs treated with TCZ as in (c) above. RIPK1 expression in IPs is shown as control. RIPK1, RIPK3 and actin expression in whole cell lysates shown below as controls. (e) RIPK1-RIPK3 complexes in indicated genotypes of T cells stimulated with anti-CD3, anti-CD28, and ZVAD for the indicated time periods. Short and long exposures of RIPK1 associated RIPK3 shown; RIPK1 and A20 expression in RIPK1 IPs are shown as controls. (f) Live cell numbers of indicated genotypes of T cells after TCR stimulation as in (e) for 72 hours. Bars indicate standard deviation. * indicates p < 0.05 when compared with WT cells. Data are representative of 3 independent experiments.