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. 2008 Sep 24;82(24):12487–12497. doi: 10.1128/JVI.00877-08

FIG. 3.

FIG. 3.

Determination of the interaction between BDV P protein and GABARAP in vivo. (A) Determination of the protein-protein interaction in 293T cells by mammalian two-hybrid analysis. 293T cells were cotransfected with pG5luc (a luciferase reporter plasmid), pVP16-P, and pGAL4-GABARAP or with control plasmids. Binding between proteins was monitored by luciferase activity, which was measured 48 h after transfection using the dual-luciferase reporter assay system. Values are expressed as the means plus standard errors of the means (three values per sample). (B) Determination of the protein-protein interaction in 293T cells by in vivo immunoprecipitations. 293T cells were cotransfected with pcDNA-P (expressing P protein) and pCMV-GABARAP (expressing Flag-tagged GABARAP). The immunoprecipitation was performed with anti-P antibody, and the Western blotting was performed with anti-Flag antibody. Lane 1, P protein alone; lane 2, P protein plus GABARAP; lane 3, extracts of untransfected 293T cells; lane 4, GABARAP alone. (C) Determination of the interaction between BDV P protein and endogenous GABARAP in 293T cells by in vivo immunoprecipitations. 293T cells were infected with BDV, and the interaction between proteins was analyzed by immunoprecipitation. In vivo immunoprecipitation was performed with anti-GABARAP antibody or anti-P antibody, and Western blotting was performed with anti-P antibody. Lane 1, 293T cells infected with BDV; lane 2, uninfected cells. (D) Determination of the interaction between BDV P protein and GABARAP in vitro. His-tagged GABARAP was bound to His-bound Sepharose beads. Some of the beads were detected by SDS-PAGE, and the rest were incubated with the extracts of BDV-infected (OL/BDV) or uninfected (OL) cells. After being washed, bound proteins were eluted with elution buffer, separated by SDS-PAGE, and probed with antibodies to P protein or anti-His antibody. Western blotting was performed with anti-P antibody. Lane 1, extracts of BDV-infected (OL/BDV) cells treated with His-GABARAP-bound agarose beads; lane 2, extracts of BDV-infected (OL/BDV) cells treated with unbound agarose beads; lane 3, extracts of uninfected (OL) cells treated with His-GABARAP-bound agarose beads; lane 4, extracts of uninfected (OL) cells treated with unbound agarose beads.