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. 2014 Jun 15;25(12):1824–1835. doi: 10.1091/mbc.E13-10-0585

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© 2014 Jacobson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — The UbE2D family of E2s promotes ubiquitination of the 26S proteasome. (A) The UbE2D family of E2s promotes ubiquitination of the 26S proteasome in vitro. Reactions were analogous to those in Figure 1C, except that each reaction contained a different E2. (B) UbE2D1 promotes ubiquitination of the 26S proteasome in cells. 293T Rpn11-HTBH cells were transiently transfected with the designated plasmids for expression of Myc-Ub or HA-UbE2D1. The 26S proteasome was purified from these cells and analyzed by immunoblotting. Overexpression of each protein was confirmed by immunoblotting of the whole-cell lysates. (C) Knockdown of UbE2D2 and UbE2D3 impairs ubiquitination of the 26S proteasome. siRNA oligos against GFP or two separate sets of oligos against UbE2D2 and UbE2D3 (a and b) were transfected into 293T Rpn11-HTBH cells, and cells were further cultured for 72 h. The 26S proteasome was then purified from these cells and analyzed by immunoblotting. Knockdown of UbE2D2 and UbE2D3 was confirmed by immunoblotting of the whole-cell lysates using an antibody recognizing both UbE2D2 and UbE2D3.