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. 2010 Dec 15;85(5):1980–1993. doi: 10.1128/JVI.01911-10

FIG. 6.

FIG. 6.

vGPCR expression induces lysine 63 TAK1 polyubiquitination. (A and B) vGPCR expression induces TAK1 polyubiquitination. HEK293T cells were transfected with 10 μg of Flag-TAK1 and/or 10 μg of vGPCR expression plasmids. (Lanes 4 to 6) Thirty-six hours posttransfection, Western blot analysis was done using TAK1 antibody (A) or the cells were subjected to TAK1 immunoprecipitation using Flag antibody followed by Western blot analysis using a ubiquitin antibody (P4D1 Ab) (B). (Lanes 1 to 3) Whole-cell extracts (WCE) reacted with P4D1 Ab. (C and D) TAK1 polyubiquitination on lysine 68. HEK293T cells were transfected with 10 μg of TAK1-Flag, along with 5 μg of the indicated ubiquitin expression plasmids with or without 10 μg of vGPCR expression plasmid. At 36 h posttransfection, total protein was extracted in lysis buffer and subjected to a Western blot experiment using Flag antibody (C, top) or vGPCR antibody (C, bottom) or subjected to Flag immunoprecipitation (TAK1 IP) followed by Western blot analysis using an HA antibody (ubiquitin Western blotting) (D).