Figure 5: Endogenous MAVS is degraded in the absence of its N-terminally truncated isoforms. | Nature Communications

Figure 5: Endogenous MAVS is degraded in the absence of its N-terminally truncated isoforms.

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

Figure 5

(a) HEK293T WT, Mavs-(M2L), Mavs-(M2&6L), Mavs-(M2-5L) and Mavs-(M2-6L) cells were subjected to subcellular fractionation to obtain P5 fractions. Various MAVS isoforms were detected by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. See also Supplementary Fig. 4a. The original full blot can be found in Supplementary Fig. 13a. (b) Whole cell lysates of HEK293T WT, Mavs-(M2L), Mavs-(M2&6L), Mavs-(M2-5L) and Mavs-(M2-6L) cells was obtained to determine endogenous MAVS protein level by immunoblotting. Tubulin was immunoblotted as a loading control. RT-PCR was performed to measure mRNA level of various Mavs gene. GAPDH was analysed as an internal control. (c–e) HEK293T WT, Mavs-(M2L), Mavs-(M2&6L), Mavs-(M2-5L) and Mavs-(M2-6L) cells were infected with VSV (MOI=1). Twelve hours post infection, RNA was extracted and qPCR was performed to measure IFN induction (c). VSV titres were quantitated by plaque assay (d). Fluorescent images were taken to examine VSV proliferation eight hours after infection (e). Scale bar represents 10 micrometres. 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. (f) HEK293T WT (lane 1–5) and Mavs-(M2-6L) (lane 6–10) cells were transfected with pcDNA3-flag-sumo-MAVS-TM, pcDNA3-flag-sumo-Bcl-xL-TM, pcDNA3-flag-sumo-VAMP-2-TM, pcDNA3-flag-sumo-Pex13-TM or empty vector. After thirty-six hours, cells were subjected to the second transfection. Cells were collected 36 h post the second transfection. Whole cell lysates were obtained to determine the endogenous MAVS protein level by immunoblotting. (g) Plasmids expressing SUMO, MAVS-TM and its mutants as indicated were transfected into HEK293T Mavs-(M2-6L) cells. Transfection was performed as described in f. Whole cell lysates were obtained to determine the endogenous MAVS protein level by immunoblotting.

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