munoprecipitation assay lysis buffer, boiled in 
1%SDS, diluted 1:10 in RIPA buffer, immunoprecipitated with antibodies specific for either RIP1, RIP2 or TRAF6, and analyzed by immunoblotting with antibodies to ubiquitin, RIP1, RIP2 or TRAF6. The whole cell lysates were directly subjected to immunoblot analysis with specific antibodies as indicated. Adenoviral-mediated Expression of A20 in Mice Decreased CVB3-induced Pro-inflammatory Cytokines Production Statistical Analysis Data were shown as the means6SEM. Statistical analysis of the data was performed using the GraphPad Prism statistical program. Means were compared using unpaired Student’s t-test. The survival rates of CVB3 infected mice were compared and analyzed with Kaplan-Meier plot. P,0.05 was considered statistically significant. A20 Alleviates Viral Myocarditis a, IL-6 and IL-1b were observed on both day 4, 7, 10 in the cardiac tissues of Ad-A20 injected mice and the expression levels of MCP-1 were significantly lower on day 4 and 7 in the Ad-A20 treated CVB3 mice. These CJ-023423 site results indicated that Ad-A20 treatment in vivo efficiently inhibited the production of pro-inflammatory cytokines in CVB3 infected mice. Treatment with Adenoviral Expressed A20 Alleviated CVB3-induced Myocarditis To investigate the therapeutic effect of A20 on CVB3-induced acute myocarditis, mice were intravenously injected with saline or 36109 pfu of either Ad-A20 or Ad-LacZ virus 2 days before CVB3 inoculation. Parameters of the severity of myocarditis, A20 Alleviates Viral Myocarditis treated with saline or Ad-LacZ developed severe myocarditis on day 7 with diffuse inflammation, whereas Ad-A20 treatment led to a significant remission of myocarditis showing few restricted mononuclear inflammation foci and tiny necrosis . All the above data indicated that Ad-A20 treatment could effectively protect mice from lethal myocarditis caused by CVB3 infection. A20 Inhibited NF-kB Signaling Activation to Suppress CVB3-induced Pro-inflammatory Cytokines Production Virus infection leads to the activation of natural immune signaling pathways. On the basis of the knowledge that nuclear factor-kappaB signaling induces transcription of various pro-inflammatory mediators, we hypothesized that A20 would inhibit NF-kB activation induced by CVB3 in vivo to restrict the inflammatory response. Mice were intravenously injected with saline or 36109 pfu of either Ad-A20 or Ad-LacZ virus 2 days before CVB3 inoculation. The cytoplasmic and nuclear protein was extracted from heart homogenates at day 4. There was a significant increase in the levels of the phosphorylation of IkBa and p65-NF-kB subunit from heart tissues of CVB3-infected mice treated with saline, which are indicators of NF-kB signaling activation, as well as an increase in the binding activity of heart nuclear extracts to a NF-kB consensus sequence compared with control mice . However, heart tissues from CVB3-infected mice treated with AdA20 showed lower levels of the phosphorylation of IkBa and p65 when compared with saline or Ad-LacZ treated group mice. The NF-kB DNA binding activity was also significantly decreased after Ad-A20 treatment. These results indicated that A20 may interfere NF-kB signaling pathway for anti-inflammation since the early stage in CVB3 induced myocarditis model. 5 A20 Alleviates Viral Myocarditis In the early stage of viral myocarditis, CVB3 infect host cardiomyocytes and trigger innate immune signaling, which is required for the induction of s