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. 2004 Jul 15;18(14):1737–1752. doi: 10.1101/gad.294904

Figure 3.

Figure 3.

Tal increases ubiquitylation of Tsg101 and affects its solubility in a RING- and PTAP-dependent manner. (A,B) HEK-293T cells were cotransfected with plasmids encoding Flag-Tsg101 (either wild type [WT] or ΔSB), HA-Tal (either wild type [WT] or H695A), and Myc-ubiquitin. Forty-eight hours after transfection, cells were divided into two portions: The first (75% of cell population) was extracted by using Triton X-100 (A), and the remainder was extracted in SDS (B). IB of immunoprecipitates or whole extracts was performed with the indicated antibodies. Oligo- and multiubiquitylated forms of Tsg101 are labeled. (C,D) HEK-293T cells transiently expressing the indicated proteins, along with Myc-Ub, were sequentially extracted in Triton X-100 (C) and SDS (D) and analyzed as indicated. (E,F) HEK-293T cells transiently expressing the indicated proteins, along with Myc-Ub, were extracted in Triton X-100 and analyzed as indicated. The TYN mutant of Tsg101 represents a protein whose TYN motif of the UEV domain (residues 67-69) has been replaced with a triplet of alanines.