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. 2016 Jan 22;7(7):8240–8252. doi: 10.18632/oncotarget.6973

Figure 4. TSGΔ154-1054 competes with TSG101 for binding Tal thereby diminishing Tal-mediated polyubiquitination of TSG101.

Figure 4

A. Binding of TSGΔ154-1054 to Tal but not TSG101. Glutathione-Sepharose-bound GST-TSGΔ154-1054 and the in vitro translated HA-Tal or TSG101 were applied to in vitro binding assay followed by western blotting. B. Binding of Tal and its deletion mutants to TSGΔ154-1054 and TSG101. A schematic illustration of the domain structures of Tal and its deletion mutants is shown (top). Tal comprises a leucine-rich repeat (LRR), ezrin-radixin-moesin (ERM) domain, coiled-coil (CC) region, sterile alpha motif (SAM), RING finger (RF), and a double PTAP motif. These HA-Tal mutants were subjected to in vitro transcription and translation, and applied to in vitro binding assay together with bead-bound GST-TSGΔ154-1054 or GST-TSG101. The result was visualized by western blotting (bottom). C. Competitive binding between TSGΔ154-1054 and TSG101 to Tal. Glutathione-Sepharose-bound GST-TSG101 and the in vitro translated HA-Tal were subjected to an in vitro competitive binding assay in the presence of increasing amounts of in vitro translated TSGΔ154-1054, and the result was revealed by western blotting (upper panel). An in vivo competitive binding between TSGΔ154-1054 and TSG101 to Tal was accomplished on TW01 cells co-transfected with HA-Tal, TSG101 and increasing level of GFP-TSGΔ154-1054 using co-immunoprecipitation coupled with western blot (lower panel). Co-immunoprecipitation was performed using anti-TSG101 antibody, and anti-GST antibody as an irrelevant antibody control. D. Effect of TSGΔ154-1054 on Tal-mediated ubiquitination of TSG101. The bead-bound GST-TSG101 was incubated with an in vitro ubiquitination reaction containing E1, E2 (UbcH5a), Ub, immunopurified HA-Tal, and in vitro translated TSGΔ154-1054. Immunoblot was performed using anti-Ub antibody.