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. 2008 Feb;19(2):754–763. doi: 10.1091/mbc.E07-09-0957

Figure 2.

Figure 2.

Tal specifically degrades Tsg101 via conjugation of polyubiquitin chains. (A) YFP-Tsg101 or YFP-Tsg101157-390 fusion proteins were coexpressed in 293T cells with decreasing amounts of YFP-Tal. Samples were lysed 24 h after transfection and analyzed by immunoblotting with an α-GFP mAb. (B) Coexpression of YFP-Tsg101 fusion protein with GST, GST-fused full-length Tal, or Tal deletions as indicated. Tal1-648 lacks the double PTAP-PSAP motif and RING domain, Tal1-674 lacks the RING domain only and TalδPTAP lacks the PTAP-PSAP motif. Twenty-four hours after transfection cells were lysed and analyzed by Western blot. YFP-Tsg101 expression was detected by immunoblotting with an α-GFP mAb and GST-Tal expression was determined using an α-Tal antibody raised against the N-terminus of Tal. (C) Comparison of GST-Tsg101 expression in the presence of YFP-Tal, YFP-Mahogunin, or empty vector control. Transfected cells were lysed 24 h after transfection and analyzed by Western blot. GST-Tsg101 or YFP- fusion protein expression was detected using α-Tsg101 and α-GFP antibodies, respectively. Lysates were probed with α-Hsp90 as a loading control. (D) Coprecipitation experiment to determine relative ubiquitination of Tsg101 by full-length or mutant Tal. 293T cells were cotransfected with plasmids encoding YFP-Tal, YFP-TalδPTAP, or empty vector with GST-Tsg101 and HA-ubiquitin. Forty-eight hours after transfection cell lysates were precipitated with glutathione-Sepharose beads, and the bound fractions were analyzed by immunoblotting with α-Tsg101 antibody. Samples were normalized for equal Tsg101 expression and probed with α-Tsg101 and α-HA antibodies. YFP-Tal and YFP-TalδPTAP expression in cell lysates was determined using α-GFP antibody. (E) Comparison of GST-Tsg101 polyubiquitination in the presence of YFP-Tal, YFP-Mahogunin or empty vector control. Cell lysates were subjected to precipitation with glutathione-Sepharose beads as before, and analyzed by Western blot. Samples were normalized for equal GST-Tsg101 expression before rerunning and probing with α-Tsg101 and α-polyubiquitin antibodies.