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Figure S4. Control for hydrolysis and reduction of ubiquitination. A complex of Ring1b and Bmi1 was subjected to in vitro ubiquitination. Ubiquitin is attached to lysine on Ring1b as shown by mass spectrometry (Buchwald, G., P. van der Stoop, O. Weichenrieder, A. Perrakis, M. van Lohuizen, and T.K. Sixma. 2006. EMBO J. 25:2465–2474). The in vitro–ubiquitinated sample without further treatment is the control (C) shown in the first lane. The other lanes show material that was recovered by precipitation with nickel-agarose beads (isolate) after it had been exposed to the indicated conditions: buffer (C), reduction with β-mercaptoethanol (βME), mild hydrolysis at pH 12, and incubation at pH 7.4 as a control. The precise procedures are outlined in Materials and methods. Recovered materials were resolved by NuPAGE and immunoblotted as indicated to detect Ring1b and its ubiquitinated species. The arrow denotes the nonubiquitinated Ring1b protein. Lower molecular mass species (bracket) are Ring1b breakdown products.