Figure 6: Spontaneously-aggregated MAVS in Mavs-(M2-6L) cells is degraded by autophagic pathway.

(a) HEK293T WT (lane 1–5) and Mavs-(M2-6L) cells (lane 6–10) were treated with DMSO or proteasome inhibitors MG132 and Bortezomib as indicated for four hours. Whole cell lysates were subjected to SDS-PAGE and immunoblotting. (b) Treatment of HEK293T WT (lane 1–5) and Mavs-(M2-6L) cells (lane 6–10) with DMSO or autophagy inhibitors 3-MA and LY294002 was performed for four hours. Whole cell lysates were used for SDS-PAGE and immunoblotting. The autophagy marker LC3 was immunoblotted. The original full blot can be found in Supplementary Fig. 13b. (c) HEK293T WT, Mavs^−/− and Mavs-(M2-6L) cells were treated with or without 30 μM chloroquine (CQ) for four hours. Whole cell lysates were used for SDS-PAGE and immunoblotting. (d–f) HEK293T WT and Mavs-(M2-6L) cells were treated with or without 10 mM 3-MA for two hours before infection with VSV (MOI=1). Twelve hours post infection, RNA was extracted and qPCR was performed to measure IFN induction (d). VSV titres were quantitated by plaque assay (e). Fluorescent images were taken to examine VSV proliferation eight hours after infection (f). Scale bar represents 10 micrometres. All data are presented as 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. (g) HEK293T Mavs^−/− cells were transfected with empty vector or plasmid expressing MAVS-(M2-6L). After 24 h post transfection, cells were infected with VSV (MOI=1) for 12 h. RNA were then extracted from collected cells and qPCR was performed to measure IFN induction. See also Supplementary Fig. 5b,c. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. (h) HEK293T WT, Mavs-(M2L), Mavs-(M2&6L), Mavs-(M2-5L) and Mavs-(M2-6L) cells were untreated or treated with 10 mM 3-MA for 4 h. Whole cell lysates were prepared for immunoblotting to determine the endogenous MAVS protein level. P5 fractions were isolated to examine MAVS aggregation.