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. 2009 Apr 17;10(5):466–473. doi: 10.1038/embor.2009.55

Figure 4.

Figure 4

Binding of ubiquitin tetramers to selected ubiquitin-binding domains. (A) Pull-down analysis with immobilized GST-tagged UBDs incubated with 1.5 μg tetraubiquitin of different linkages. Three lanes per linkage correspond to 5% of input tetraubiquitin, GST–UBD-bound tetraubiquitin and GST control-bound tetraubiquitin. Ponceau-stained membranes are shown in supplementary Fig 3 online. (B) Pull-down analysis as in (A), in which the different tetraubiquitins were mixed and used as inputs. The Ponceau-stained membrane shows the GST–UBDs. (C) The input samples from (B; first four lanes) were silver and Coomassie stained, which indicated the antibody does not recognize different linkages equivalently. (D) Potential mechanisms of UBD binding to differently linked polyubiquitin chains. (E) Summary of specificity for all proteins tested. Full-length TRABID is further analysed in supplementary Fig 4 online. ABIN2(FL), A2O binding inhibitor of NF-κB signalling 2 (full length); AMSH, associated molecule with the SH3 domain of STAM; CARD, caspase recruitment domain; cIAP1, cellular inhibitor of apoptosis 1; DUBs, deubiquitinases; GST, glutathione S-transferase; IB, immunoblotting; JAMM, JAB1/MPN/Mov34; MUD1, UBA domain-contining protein mud1; NZF, Npl4 zinc finger; OTU, ovarian tumour; TAB2, TAK1 binding protein 2; Ub, ubiquitin; UBA, ubiquitin associated; UBAN, ubiquitin binding in ABIN and NEMO; UBD, ubiquitin-binding domain; UCH, ubiquitin carboxy-terminal hydrolase; USP, ubiquitin specific protease.