Figure 1: N-terminally truncated isoforms of MAVS were separated from full-length MAVS upon virus infection. | Nature Communications

Figure 1: N-terminally truncated isoforms of MAVS were separated from full-length MAVS upon virus infection.

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

Figure 1

(a) A diagram illustrating that six MAVS isoforms, including MAVS- M1/2/3/4/5/6, are translated from the polycistronic Mavs transcript. (b) Immunoblotting showing that six MAVS isoforms are expressed in HEK293T cells. P5 fractions containing mitochondria were obtained from HEK293T cells treated with or without siRNA oligoes targeting Mavs (si-MAVS) and subjected for immunoblotting (Lane 1, 2). Lane 3–8, HEK293T Mavs−/− cells were transfected with constructs expressing Flag-tagged MAVS-M1/M2/M3/M4/M5/M6, respectively. Whole cell lysates were then prepared at thirty-six hours post transfection and used for SDS-PAGE and immunoblotting, to indicate the migration position of the ectopically expressed MAVS isoforms in Lane 1 and 2. Mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control. Endogenous MAVS isoforms were indicated as arrows. Non-specific bands were labelled with asterisks. The original full blot can be found in Supplementary Fig. 11a. (c) Multiple isoforms of MAVS were detected in various human cell lines. P5 fractions from HEK293T, HeLa, A549 and THP-1 were obtained and subjected to immunoblotting with anti-MAVS-(C) antibody (Lane 1–4). Prohibitin was immunoblotted as an internal control. As described in (b lane 3–8), Flag-tagged MAVS isoforms were immunoblotted to indicate the migration position of MAVS truncated forms (Lane 5–10). Endogenous MAVS isoforms were indicated as arrows. Non-specific bands were labelled with asterisks. See also Supplementary Fig. 1b. The original full blot can be found in Supplementary Fig. 11b. (d) HEK293T cells infected with or without vesicular stomatitis virus (VSV) for 12 h were collected and subjected to subcellular fractionation. P5 fractions containing mitochondria were collected and solubilized with n-Dodecyl-β-D-maltoside (DDM) before sucrose gradient centrifugation. Nine fractions from top to the bottom were taken and analysed by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. MAVS activities in stimulating IRF3 dimerization were also examined. Arrows indicate the peaks of standard proteins (160 and 450 kDa) and blue dextran 2,000 (2,000 kDa) used as molecular weight markers. Asterisk indicates a non-specific band. The original full blot can be found in Supplementary Fig. 11c.

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