Fig. 4
From: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
MLL5 deficiency results in accumulation of RIG-I protein. a Luciferase activity of ISRE promoter reporter in HEK293T cells transfected with expression plasmids for MAVS-FLAG (200 ng), TBK1-FLAG (200 ng) or IRF3-HA (200 ng) along with MLL5-FLAG (600 ng) or empty control vector (600 ng) for 24 h. Results were presented relative to the luciferase activity in control cells (transfected with luciferase reporter and empty vector). Immunoblot analysis of MAVS-FLAG, TBK1-FLAG, MLL5-FLAG or IRF3-HA are shown below. Actin served as a loading control. b, c Immunoblot analysis of MLL5, RIG-I, MDA5, and MAVS in WT and MLL5−/− HEK293T cells (b) or in BMDMs from WT and Mll5−/− mice (c). d Immunoblot analysis of MLL5 and RIG-I in WT and Mll5−/− MEFs with RIG-I knockdown. Actin served as a loading control. e Expression of Ifn-β and Tnf-α mRNA in WT and Mll5−/− MEFs with RIG-I knockdown infected with VSV-GFP (MOI:0.5) and HSV-1 (MOI:1) for 6 h. Gapdh served as a control. f Expression of DDX58 mRNA in WT and MLL5−/− HEK293T cells treated with actinomycin D (1 μg/ml) for indicated times. GAPDH served as a control. g, h Immunoblot analysis of RIG-I in WT and MLL5−/− HEK293T cells treated with CHX (100 μg/ml) (g) or CHX (100 μg/ml) and MG132 (30 μM) (h). Actin served as a loading control. Relative band densities indicating protein levels are shown blown each band (g). Quantification of relative RIG-I protein levels is shown in the right panel. Band density indicating protein amount was quantified using Image J software. i Co-immunoprecipitation and immunoblot analysis of K48-linked polyubiquitination of RIG-I in WT and MLL5−/− HEK293T cells transfected with RIG-I-FLAG. j His-pull down and immunoblot analysis of K48-linked polyubiquitination of RIG-I in WT and MLL5−/− HEK293T cells cotransfected with RIG-I-FLAG and mutant ubiquitin K48-ubi-His. k Co-immunoprecipitation and immunoblot analysis of K48-linked polyubiquitination of RIG-I in HEK293T cells cotransfected with FLAG-RIG-I and MLL5-HA plasmids (0, 300, and 1000 ng). The HEK293T cells were treated with MG132 (5 μM) for 12 h before harvest (i–k). Data were representative of three independent experiments (b–d, g–k) or were from three independent experiments (a, e, f) and were analyzed by Student’s t-test (two-tailed) and were presented as mean ± SD
