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. Author manuscript; available in PMC: 2014 Mar 15.
Published in final edited form as: Oncogene. 2013 Apr 1;33(11):1438–1447. doi: 10.1038/onc.2013.78

Figure 4. Pin1 prevents E6AP-mediated in vitro ubiquitination of ERα.

Figure 4

a) Purified recombinant ERα and GST or GST-E6AP were allowed to form complexes for 1 h, and then ERα was immunoprecipitated using ERα antibody or mouse IgG antibody and Western blot performed for GST, pS118ERα, and ERα.

b) ERα was in vitro ubiquitinated as described in the experimental procedures with Ube1, UbcH5a, ATP, ubiquitin, and GST-E6AP (0–0.6 µg) at 30°C for 1.5 h, and Western blot was performed for ERα.

c) ERα was in vitro ubiquitinated as in (b) with and without 0.4 µg GST-E6AP in the presence and absence of Pin1 (0–1 µg), and Western blot was performed for ERα.

d) ERα was ubiquitinated as in (c) with and without GST-E6AP and Pin1 in the presence and absence of juglone (7.5 µM), and Western blot was performed for ERα.