Fig 1: NS1 recruits MMP-9 to interact with adhesion and tight junction proteins.(A) HEK293T cells were transfected with plasmid encoding HA-NS1 plus Flag-β-catenin. Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-Flag, anti-HA, or anti-β-catenin antibody. Cell lysates (40 μg) were used as Inputs. (B) Hela cells were transfected with plasmid encoding HA-NS1, Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-HA or anti-β-catenin antibody. Cell lysates (40 μg) was used as Input. (C, D) HEK293T cells (C) or Hela cells (D) were co-transfected with plasmid encoding HA-NS1 plus Flag-ZO-1, Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-Flag or anti-HA antibody. Cell lysates (40 μg) were used as Inputs. (E, F) HEK293T cells were transfected with Flag-β-catenin (E) or Flag-ZO-1 (F) and incubated with purified His-SARS-CoV-2-N (5 μg) or His-DENV2-NS1 (5 μg) for 24 h, cell extracts were incubated with Ni-NTA Agarose beads. Mixtures were analyzed by immunoblotting using anti-β-catenin, anti-NS1, anti-His, anti-ZO-1 antibody. Untreated proteins including His-SARS-CoV-2-N (1 μg) and His-DENV2-NS1 (1 μg) and HEK293T cell lysates were analyzed by immunoblotting using anti-β-catenin, anti-NS1, anti-His, and anti-ZO-1 antibody (as input). (G, H) Hela cells were co-transfected with plasmid encoding HA-NS1 plus Flag-MMP-9, Cell lysates were immunoprecipitated using anti-Flag (G) or anti-β-catenin antibody (H), and analyzed using anti-Flag, anti-HA or anti-β-catenin antibody. Cell lysates (40 μg) was used as Input. (I, J) Hela cells were co-transfected with plasmid encoding HA-NS1, HA-MMP-9, and Flag-ZO-1 (I) or Flag-NS1, HA-MMP-9, and Flag-ZO-1 (J). Cell lysates were immunoprecipitated using anti-Flag (top) or anti-HA antibody (bottom), and analyzed using anti-Flag, anti-HA or anti-MMP-9 antibody. Cell lysates (40 μg) was used as Input. (K, L) HUVEC cells were treated with NS1 protein (5 μg/ml), MMP-9 protein (100 ng/ml), and NS1 protein (5 μg/ml) plus MMP-9 protein (100 ng/ml), respectively, for 6 h. The distributions of the membrane marker (Dil) (yellow), the endogenous β-catenin (yellow) and ZO-1 (yellow) proteins and the extracellular NS1 (red) and MMP-9 (green) proteins were visualized under confocal microscope. The quantifications of co-localization fluorescence were determined by ImageJ software (L). ND means not detected. All dates were representative of three independent experiments.