Fig 1: Ube2N is required for RIG-I and MAVS activation in MEFs.(a–c) Small hairpin RNAs (sh-Ube2D1/2/3/4 & N) as indicated was transduced into MEFs with lentivirus. Eighty four hours after transduction, the cells were infected with VSV. Sixteen hours post infection, the cells were collected and IFN β (a) and IFN α (b) production were measured by qPCR. Immunoblotting were also performed to examine knockdown efficiency (c). *P<0.05 and ***P<0.001. NS indicates no statistically significant difference. (d) MEFs were transduced with or without sh-Ube2N. Eighty four hours after transduction, Flag-MAVS was further transduced into MEFs by retrovirus for forty eight hours. The cells were then infected with VSV. Sixteen hours post infection, the cells were collected to measure cytokine production by qPCR. See also Supplementary Fig. 4d. (e) MEFs were treated as described in a except that the cells were collected to isolate P5 fractions. P5 fractions were subjected to SDD-AGE to examine MAVS aggregation and IRF3 dimerization assay in vitro. The original full blot can be found in Supplementary Fig. 9a. (f) MEFs were treated as described in d except that Flag-Ube2D3 or Flag-Ube2N but not Flag-MAVS was transduced into the cells by retrovirus as indicated. See also Supplementary Fig. 4e.
Fig 2: SHPRH recruits UBE2D1 to, and forms stable complexes with, nucleosomes. a Western blot of SHPRH and UBE2D1 immunoprecipitations. FLAG-tagged versions of SHPRH and UBE2D1 were overexpressed in HEK293 cells. Whole cell extracts were then used to immunoprecipitate the two proteins using specific antibodies, as indicated on top. IgG was used as a negative control. The input (IN), the flow-through (FT) and the immunoprecipitates (IP) were probed using antibodies (α) against SHPRH and UBE2D1, as indicated on the right. b Electrophoretic mobility shift assay. SHPRH (20 nM) was incubated with radiolabeled mononucleosomes (20 nM; lane 2), or, in addition, with UBE2D1 (800 nM; lane 3). As controls, we used mononucleosomes alone (20 nM; lane 1) or incubated in the presence of UBE2D1 or BSA (800 nM; lanes 4 and 6, respectively) or, SHPRH + BSA (lane 5), as indicated on top. Reactions were separated by native PAGE and visualized using a phosphorimager screen. The positions of free (unbound) nucleosomes and protein-nucleosome complexes are indicated by arrows, on the right
Fig 3: UBE2D1 and UBE2D2 bind to VEGFR2 and promote downregulation in endothelial cells. (A) Immunoblot analysis of basal VEGFR2 levels in primary endothelial cells following mock proteofection or proteofection of human recombinant UBE2D1 or UBE2D2 protein for 3 h. Antibodies to VEGFR2, UBE2D1 and UBE2D2 (polyclonal anti-UBE2D1 used to detect both UBE2D1 and UBE2D2, indicated as UBE2D1/2), PECAM-1, alkaline phosphatase (AP) and tubulin (control) were used to analyse protein levels. The three visible bands for VEGFR2 represent immature non-glycosylated (lower band), partially glycosylated (middle band) and mature fully glycosylated (upper band) forms. All forms were quantified together. (B) Quantification of immunoblot data of relative VEGFR2 levels in endothelial cells after mock proteofection (control) or proteofection with UBE2D1 or UBE2D2 recombinant protein. Error bars indicate ±s.e.m. Significance was determined by one-way ANOVA followed by Tukey's post-test for multiple comparisons and indicated by asterisks (n=4). *P<0.05.
Fig 4: UBE2D1 and UBE2D2 regulate VEGF-A-stimulated endothelial tubulogenesis. (A) Analysis of tubule formation using a fibroblast-endothelial co-culture assay after transfection of primary endothelial cells with control non-targeting, UBE2D1 or UBE2D2 siRNA for 72 h in the presence or absence of VEGF-A. Tubules were marked using anti-PECAM-1. Scale bar: 400 μm. (B–D) Quantification of tubulogenesis data for (B) total length, (C) total size and (D) total number of branch points. Three images per condition were analysed for each of three independent experiments. Error bars indicate ±s.e.m. Significance was determined by two-way ANOVA followed by Dunnett's post-test for multiple comparisons (n=3). ns, not significant; *P<0.05; **P<0.01.
Fig 5: VEGFR2 levels remain elevated after E2 knockdown despite inhibition of synthesis of new proteins. (A) Immunoblot analysis of phosphorylated and total VEGFR2 levels after E2 knockdown for 72 h and treatment with cycloheximide for the indicated durations to prevent new protein synthesis, with transferrin receptor (TfR) levels immunoblotted as control. The triple bands observed for VEGFR2 represent immature non-glycosylated (lower band), partially glycosylated (middle band) and fully glycosylated mature (upper band) forms. (B) Quantification of immunoblot data of relative VEGFR2 levels after UBE2D1 and UBE2D2 knockdown and cycloheximide treatment. Significance was determined by two-way ANOVA followed by Dunnett's post-test for multiple comparisons and indicated by asterisks (n=3). (C) Analysis of basal VEGFR2 in primary endothelial cells after E2 knockdown for 72 h and cycloheximide (CHX) inhibition of new protein synthesis for the indicated durations as measured by immunofluorescence microscopy to detect VEGFR2 (green) or nuclear DNA (blue). Scale bar: 50 µm. (D) Quantification of immunofluorescence data of relative VEGFR2 levels in primary endothelial cells after control non-targeting, UBE2D1 or UBE2D2 siRNA treatment for 72 h and cycloheximide treatment for the indicated durations (see Materials and Methods). Relative VEGFR2 levels were quantified as described in the legend of Fig. 1A. Error bars indicate ±s.e.m. Significance was determined by two-way ANOVA followed by Dunnett's post-test for multiple comparisons (n=3). *P<0.05; **P<0.01; ***P<0.001.
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