EV71 2A^pro cleaves MAVS.

<p>(<b>A, B</b>) <i>In vitro</i> dose- dependent cleavage assay of EV71 2A^pro (A) and 3C^pro (B) on MAVS using LI-COR Odyssey Dual-Color System. Increasing doses of recombinant proteases were added to the cell lysates and incubated at 37°C for 6 h (from 0–200 ng/µL, lanes 2–8); recombinant proteases (lane 1) and EV71-infected HeLa cells (lanes 9–12) served as negative and positive controls, respectively. Two antibodies recognizing different MAVS epitopes were used (E-3, 700 nm, green; AT107, 800 nm, red). An overlay of the two channels is shown in the “Merge” panel. White arrows indicate cleaved bands in EV71-infected HeLa cells and the same-size bands in 2A^pro-cleaved HeLa extracts. (<b>C</b>) Western blot analysis of MAVS in HeLa cells transfected with increasing doses of plasmids (0–4 µg) encoding EV71 3C^pro (lanes 1–4) and 3ABC (lanes 5–8) precursor protein fused with GFP. The MAVS image is an overlay of two signals from the different channels described in (A). The same cell lysates were also used to detect 3C^pro and 3ABC using an antibody against GFP; actin served as the loading control. (<b>D</b>) Western blot analysis of MAVS in BSRT7/5 cells transfected with increasing doses of pcDNA3.1-IRES-2A plasmid (lanes 1–5, 0–4 µg) and pcDNA3.1-EGFP control plasmid (4 µg). (<b>E</b>) <i>In vitro</i> dosage cleavage assay (0–200 ng/µL) of 2A^pro (lanes 2–8) and mutated 2A^pro (2A^pro-110) (lanes 10–16) on MAVS; recombinant 2A^pro (lane 1) and 2A^pro-110 (lane 9) served as negative controls. The MAVS image is an overlay of two signals from the different channels described in (A). PABP was used as a readout for the enzyme activity of 2A^pro and 2A^pro-110; actin served as a loading control. The image of 2A^pro and 2A^pro-110 (lower panel) is a direct scan of SDS-PAGE gel stained with Coomassie brilliant blue.