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. 2015 Sep 21;29(11):1646–1657. doi: 10.1210/me.2015-1125

Figure 3. Estrogen enhances the E3 ligase activity of UBE3C in the presence of ERα.

Figure 3.

A, Recombinant FLAG-UBE3C was incubated with ubiquitin, E1, and the indicated E2s (UBE2s, 83nM each) in the presence of ATP. The reactions were immunoblotted with antiubiquitin antibody. B, The ubiquitin ligation reaction was performed as in A with UBE2D3 and in the absence or presence of an increased amount of UBE3C. C, The ubiquitin ligation reaction was performed as in A with UBE2D3, UBE3C (13.3μM), and the indicated amount of ERα (shown as mole ratios to UBE3C) and 17β-estradiol (lanes 2, 5, and 8: 10nM; lanes 3, 6, and 9: 10μM). The reactions were immunoblotted with the indicated antibodies. *, nonspecific product from ERα preparation. D, The ubiquitin ligation reaction was performed as in C with or without 10nM 17β-estradiol and an increased amount of 4-hydroxytamoxifen (4OHT) (lanes 3 and 6, 100nM; lanes 4 and 7, 1μM; and lanes 5 and 8, 10μM). *, nonspecific product.