Fig 2.
MAVS is cleaved in EV71-infected cells.
Western blot analysis of MAVS expression in EV71-infected (MOI = 10) (A) HeLa cells and (B) RD cells for the indicated time. The time course evaluating MAVS expression was carried out by LI-COR Odyssey Dual-Color System using two different antibodies against MAVS (E-3, 700 nm, green; AT107, 800 nm, red). Results are displayed as images from each channel as well as an overlaid image of the two channels. Arrows indicate full-length MAVS (FL) and the cleaved fragments (CF) derived from MAVS.
Fig 7.
(A, B) In vitro dose- dependent cleavage assay of EV71 2Apro (A) and 3Cpro (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 2Apro-cleaved HeLa extracts. (C) Western blot analysis of MAVS in HeLa cells transfected with increasing doses of plasmids (0–4 µg) encoding EV71 3Cpro (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 3Cpro and 3ABC using an antibody against GFP; actin served as the loading control. (D) 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). (E) In vitro dosage cleavage assay (0–200 ng/µL) of 2Apro (lanes 2–8) and mutated 2Apro (2Apro-110) (lanes 10–16) on MAVS; recombinant 2Apro (lane 1) and 2Apro-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 2Apro and 2Apro-110; actin served as a loading control. The image of 2Apro and 2Apro-110 (lower panel) is a direct scan of SDS-PAGE gel stained with Coomassie brilliant blue.
Fig 9.
In vitro cleavage assay of EV71 2Apro on MAVS and MAVS mutants expressed in HeLa cells.
(A) The cell lysates from stable cell lines expressing WT-MAVS (lanes 1&2), m-MAVS-3M (lanes 3&4), m-MAVS-209 (lanes 5&6), m-MAVS-251 (lanes 7&8), and m-MAVS-265 (lanes 9&10) were incubated with 200 ng/µL 2Apro at 30°C for 2 h; the cell lysates were then subjected to western blot analysis to probe MAVS using an HA antibody against an HA peptide fused to the C-terminus of MAVS and MAVS mutants. Cleavage of PABP served as a readout for the enzymatic activity of 2Apro. (B) Schematic diagram analyzing cleavage results from (A). The differing line thickness represents the differing extent of cleavage activity of 2Apro on each substrate. (C, D, E) Time-course study of 2Apro on WT-MAVS (C), m-MAVS-251, (D) and m-MAVS-265 (E) stably expressed in their corresponding cell lines by western blot, which was carried out with 200 ng/µL recombinant 2Apro at 30°C for indicated time.