Figure 3: MAVS-TM inhibits aggregation of endogenous MAVS through a specific homotypic interaction. | Nature Communications

Figure 3: MAVS-TM inhibits aggregation of endogenous MAVS through a specific homotypic interaction.

From: Multiple truncated isoforms of MAVS prevent its spontaneous aggregation in antiviral innate immune signalling

Figure 3

(a) A diagram showing SUMO tagged MAVS-TM, Bcl-xL-TM, VAMP-2-TM, Pex13-TM. (b) pcDNA3-HA-MAVS was transfected into HEK293T cells together with pcDNA3-flag-sumo, pcDNA3-flag-sumo-MAVS-TM, pcDNA3-flag-sumo-Bcl-xL-TM, pcDNA3-flag-sumo-VAMP-2-TM, or pcDNA3-flag-sumo-Pex13-TM. Thirty-six hours post transfection, cells were collected and subjected to immunoprecipitation. The original full blot can be found in Supplementary Fig. 12b. (c) Empty vector or plasmids encoding various TMs as described in (b) were transfected into HEK293T cells together with IFN-luciferase reporter. Cells were infected with Sendai virus twenty-four hours after transfection, and IFN induction were measured twelve hours post infection by luciferase reporter assay. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t-test. ***P<0.001. N.S. indicates no statistically significant difference. (d) Experiments were performed as described in (c) except that IFN-luciferase reporter was omitted. Cells were collected and P5 fractions were isolated to examine MAVS aggregation. Whole cell lysates were subjected to SDS–PAGE and immunoblotting. (e) pcDNA3-HA-MAVS was transfected into HEK293T cells together with pcDNA3-flag-sumo-MAVS-TM, pcDNA3-flag-sumo-TM-(A521W), pcDNA3-flag-sumo-TM-(G524W), pcDNA3-flag-sumo-TM-(L530W) or pcDNA3-flag-sumo-TM-(L534W). Thirty-six hours post transfection, cells were collected and subjected to immunoprecipitation with HA beads and following immunoblotting. (f) Empty vector or constructs encoding MAVS-TM and various mutants as described in (e) were transfected into HEK293T cells together with IFN-luciferase reporter. Cells were infected with Sendai virus twenty-four hours after transfection, and IFN induction were measured twelve hours post infection by luciferase reporter assay. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t-test. **P<0.01, ***P<0.001. N.S. indicates no statistically significant difference.

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