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. 2002 Jun;76(11):5701–5710. doi: 10.1128/JVI.76.11.5701-5710.2002

FIG. 4.

FIG. 4.

LGT virus NS3 protein binds to human caspase-8. 293T cells were transfected with plasmids expressing F-NS3 and Casp8m-HA, Casp2m-HA, Casp3m-MYC, or Casp9m-MYC, as indicated. At 18 h after transfection, cell lysates were incubated with mouse anti-HA.11 (lanes 1 to 3) or anti-MYC (lanes 4 and 5) MAb and immunoprecipitated by anti-mouse IgG-conjugated Sepharose beads. Bound proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to a membrane, and probed with anti-Flag MAb to detect coprecipitating F=NS3 (A). Expression of the epitope-tagged proteins in cells was confirmed by immunoblotting aliquots of cell lysates with anti-Flag (B), anti-HA.11 (C), or anti-MYC (D) MAb. (E) 293T cells were transfected with plasmids expressing epitope-tagged proteins, and cell lysates were immunoprecipitated with anti-Flag MAb. Immunoblotting was performed with anti-caspase-8 (lanes 1 and 2), anti-HA.11 (lanes 3 and 4), anti-caspase-3 (lanes 5 and 6), and anti-caspase-9 (lanes 7 and 8) MAb. Caspases are indicated by asterisks. (F) LGT virus NS3 immunoprecipitates with wild-type caspase-8. 293T cells were transfected with plasmids expressing F-NS3 and Casp2-HA or Casp8-HA. Cell lysates were precipitated with the anti-HA.11 antibody, and precipitates were blotted with the mouse anti-Flag antibody.