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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 2. Direct activation of TAK1 by unanchored polyubiquitin chains.

Figure 2

a, Ubiquitination reaction was carried out in the presence of E1, Ubc13/Uev1A, TRAF6 and ubiquitin, then E1 and E2 were inactivated with NEM (step 1) before incubation with the TAK1 complex in the presence of ATP to measure TAK1 activation (step 2). b, Ubiquitination reactions were carried out as in step 1 in (a), then incubated with TAK1 and its substrate MKK6(K82A). c, Ubiquitination mixtures were incubated with nickel-NTA beads, then the bound and unbound fractions were tested for stimulation of TAK1. d, Top: procedure for purification of TAK1 activator; bottom: the Mono S fractions were analyzed for TAK1 activation and by silver staining and immunoblotting. e, Different concentrations of purified polyubiquitin chains (Mono S fraction 15 in panel d) were tested for TAK1 activation. f, K63- and K48-linked polyubiquitin chains were tested for TAK1 activation.