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. 2020 Feb 4;11:42. doi: 10.3389/fmicb.2020.00042

FIGURE 6.

FIGURE 6

Non-structural protein 3D binds RIPK3. (A) Non-structural protein 3D in 3D-transfected cells was evaluated by Western blotting before or after immunoprecipitation (IP) with RIPK3 antibody (Santa) at 36 h post transfection. Results are representative of three independent experiments. (B) Non-structural protein 3C in 3C-transfected cells was evaluated by Western blotting before or after immunoprecipitation (IP) with RIPK3 antibody (Santa) at 36 h post transfection. Results are representative of three independent experiments. (C) The expression of RIPK3 in 293T cells was analyzed at 36 h after transfection with 0, 1, or 2 μg of 3D-HA-VR1012 plasmid as indicated. Results are representative of three independent experiments. Histone is loading control. (D) Transfection of 0, 1, or 2 μg of 3D-HA-VR1012 plasmid into RD cells (HA-VR1012 plasmid was used to replenish 2 μg/well) for 36 h, cell morphologic analysis with propidium iodide staining by light microscopy (left) or fluorescence microscopy (right). Results are representative of three independent experiments. Bar = 20 μm. (E) The localization of RIPK3 and HA was analyzed by fluorescence microscopy at 48 h post-transfection of VR1012-HA and VR1012-3D-HA. Cells were fixed in 4% paraformaldehyde, stained with RIPK3 and HA antibodies, and then co-stained FITC labeled-secondary antibody (Green, HA) and CoraLite594-conjugated-second antibody (Red, RIPK3). Merged images of HA and RIPK3 are shown. Data are representative of three individual experiments. Scale bar = 10 μm.