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. Author manuscript; available in PMC: 2009 Jul 21.
Published in final edited form as: Oncogene. 2006 Mar 2;25(9):1330–1339. doi: 10.1038/sj.onc.1209167

Figure 3. TSSC5 ubiquitylation.

Figure 3

(a) TSSC5 C-terminus is polyubiquitylated in vivo. 293T cells were transfected with plasmid encoding HA-tagged TSSC5 C-terminus (TSSC5C). After 18 h, cells were incubated with or without 10 µm MG132 for 2 h. Cell extracts were denatured to break up protein complexes and then HA-tagged proteins were immunoprecipitated with anti-HA antibody. The immunoprecipitates were blotted with anti-HA or antiubiquitin. (b) TSSC5 6th loop is the smallest fragment that is polyubiquitylated efficiently in vivo. Plasmid encoding HA-tagged TSSC5 6th loop was transfected into 293T cells. At 18 h after transfection, cells were treated with MG132 for 2 h and then extracts were prepared for immunoprecipitation with anti-HA antibody in denaturing conditions. Immunoprecipitates were blotted with anti-HA and anti-ubiquitin. Only fragments of TSSC5 containing the 6th loop exhibited polyubiquitylation, while other regions containing lysines (such as the TSSC5 10th loop) did not exhibit polyubiquitylaion (negative data, not shown). (c) Only the combination of UbcH6 and RING105 facilitated polyubiquitylation of TSSC5C in vitro. Immunoprecipitated GFP- or GFP-RING105 was incubated with E1, the indicated E2, GST-ubiquitin, ATP and S-tagged TSSC5C for 2 h at 30°C. Reactions were terminated with 1% SDS and S-tagged protein was collected with S-protein beads. Ubiquitylation was probed with anti-GST. Although several of the E2’s result in the accumulation of high molecular weight bands on the S-tag-precipitated protein, only UbcH6 results in bands that are generated only in the presence of RING105. (d) More detailed examination of the UbcH6/RING105-mediated ubiquitylation reaction shows that high molecular weight bands contain both TSSC5 (labeled with anti-S tag) and GST-ubiquitin (labeled with anti-GST), demonstrating that the high molecular weight bands are poly(GST)ubiquitylated forms of TSSC5C. Note that in the presence of RING105, majority of TSSC5 was polyubiquitylated. In contrast, without RING105 polyubiquitin chain was rarely observed.