Fig. 5

Rnf19a regulates JEV-induced inflammatory response in BV2 cells by mediating RIG-I degradation. A HEK-293T cells were co-transfected with luc-NF-κB reporter, pRL-TK construct together with pCAGGS-Flag-TRAF6 plasmid, pCAGGS-Rnf19a plasmid or pCAGGS empty vector. The luciferase activity was determined, and the expression of indicated proteins was detected by immunoblot assay at 36 h post-transfection. B HEK-293T cells were co-transfected with the luc-NF-κB reporter, pRL-TK construct and pCAGGS-Flag-RIG-I N plasmid, pCAGGS-Rnf19a plasmid or pCAGGS empty vector. The luciferase activity was determined and the expression of indicated proteins was detected by immunoblot assay at 36 h post-transfection. C BV2 cells were infected with JEV at an MOI of 5. At 12, 24 and 36 hpi, the expression of RIG-I and Rnf19a were determined by immunoblotting (left panel), and quantified by immunoblotting scanning using Image J software and normalized to the amount of GAPDH (right panel). D BV2 cells were transfected with pCAGGS-Rnf19a plasmid or pCAGGS empty vector. The protein level of RIG-I was determined by immunoblot assay at 36 h post-transfection. E HEK-293T cells were co-transfected with pCAGGS-Flag-RIG-I plasmid and pCAGGS-Rnf19a plasmid or pCAGGS empty vector. At 24 h post-transfection, cells were treated with DMSO, the proteasome inhibitor MG132 (10 μM) or the lysosome inhibitor NH₄Cl (10 mM). After incubation for 4 h, the protein levels of RIG-I and Rnf19a were determined by immunoblot assay. F HEK-293T cells were transfected with pCAGGS-Flag-RIG-I plasmid along with pCAGGS empty vector or pCAGGS-Rnf19a. The cell lysates were subjected to denaturing immunoprecipitation with anti-Flag beads and the products were analyzed by immunoblotting with the indicated antibodies. G HEK-293T cells were co-transfected with pCAGGS-Flag-RIG-I plasmid, pCAGGS empty vector or a pCAGGS-Rnf19a construct and plasmid encoding HA-WT-ubiquitin (Ub). Followed by treatment with MG132, cells lysates were subjected to denaturing immunoprecipitation with anti-Flag beads and products were analyzed by immunoblotting with indicated antibodies. H BV2 cells were co-transfected with pCAGGS-Rnf19a plasmid and pCAGGS-Flag-RIG-I N plasmid or pCAGGS empty vector following JEV infection at an MOI of 5. At 36 hpi, the mRNA abundance of inflammatory cytokines was measured by qRT-PCR. I Rnf19a knockdown (Rnf19a KD) or negative control (NC) BV2 cells were treated with GSK343 (10 μM) or equal volume of DMSO following JEV infection at MOI of 5. Data are expressed as means ± SEM from three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001, NS represents no significant difference