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. 2022 May 23;96(12):e00317-22. doi: 10.1128/jvi.00317-22

FIG 9.

FIG 9

Full-length FMDV 3A protein impaired ANXA1-TBK1 complex formation. (A) Diagrammatic sketch of 3A and its mutants containing a GFP tag. (B) Plasmids of ANXA1 and full-length 3A or its mutants with GFP tag were transfected into HEK293T cells. After 24 h, the cell lysates were subjected to the co-IP assay, and anti-myc monoclonal antibody was used as the IP primary antibody. (C) Plasmids of 3A-GFP, Flag-3A (aa 93 to 102, Δ93-102aa), Flag-3A (Δ87-106aa), or Flag-3A (E78G, H80C, K84 N, A97P, and R127I of 5-aa-site mutant, Δ5aa) deletion together with myc-ANXA1 were transfected into HEK293 cells. After 24 h, cell lysates were subjected to IP detection. IgG, Flag, or GFP was used as IP primary antibody. (D and E) Plasmids of FMDV 3A-GFP or EV together with myc-ANXA1 or EV were transfected into HeLa cells. (D) After 24 h, the cells were fixed and subjected to confocal imaging, and the distribution of ANXA1 in the cells was analyzed. (E) Thirty cells were observed, and the number of cells in which ANXA1 was dispersed in the cytoplasm and nucleus and the number of cells in which ANXA1 was distributed around the membrane was statistically analyzed. (F) Plasmids of myc-ANXA1 and Flag-TBK1 and GFP or 3A-GFP were transfected into HEK293 cells. After 24 h, the cell lysates were subjected to the co-IP assay, and anti-myc monoclonal antibody was used as IP primary antibody. (G) Plasmids of His-ANXA1 or His-TBK1 were transferred into bacteria of Escherichia coli (BL-21), and the proteins were purified. EV or 3A-GFP was transfected into HEK293 cells. After 24 h, the cell lysates were collected. Next, the same concentration of EV or 3A cell lysate was added into ANXA1 and TBK1 mixture at 4°C for 4 h. The mixtures were subjected to the pulldown assay, and anti-ANXA1 monoclonal antibody was used as the pulldown primary antibody. (H) Plasmids of 3A (500 ng) plus EV (500 ng), ANXA1 (500 ng) plus EV (500 ng), or ANXA1 (500 ng) plus 3A (500 ng) were transfected into PK15 cells. After 24 h, the cells were infected with FMDV at indicated time points. Cellular RNA was extracted and reverse transcribed into cDNA. The relative mRNA level was detected by qPCR. Data are representative of three independent experiments. The data are expressed as means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA; GraphPad Prism 8.3.0).