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. Author manuscript; available in PMC: 2010 Mar 3.
Published in final edited form as: Nature. 2009 Aug 12;461(7260):114–119. doi: 10.1038/nature08247

Figure 4. Regulation of IKK by unanchored polyUb chains and CYLD.

Figure 4

a. Ubiquitination reactions containing TRAF6 and Ubc13/Uev1A or Ubc5 were analyzed by immunoblotting with the indicated antibodies. b. Aliquots of reaction mixtures from (a) were denatured by heating in 1% SDS, then diluted to 0.1% SDS before immunoprecipitation with a TRAF6 antibody. c, PolyUb synthesized in the presence of TRAF6 and Ubc5 were pre-incubated with isopeptidase T or its mutant C335A, then incubated with the IKK complex and IκBα to measure IKK activation. d. Polyubiquitinated TRAF6 (Ubn-TRAF6) was synthesized in the presence of Ubc5, then incubated with the indicated DUBs before immunoblotting. e, Linear polyubiquitination of NEMO (Ubn-NEMO) was carried out in the presence of Ubc5 and HOIL-1L/HOIP (E3), then Ubn-NEMO was incubated with the indicated DUBs followed by immunoblotting.