Welcome to Gaia! :: View User's Journal | Gaia Journals

For the Luciferase assays [19], N6022 at 40–50% confluence were transfected with the indicated plasmids [0.25 μg reporter plasmids, 0.025 μg ERα, 0.1 μg ERαΔD/E/F, AhR/Arnt (+, 0.05 μg; ++, 0.1 μg; +++, 0.2 μg), 0.1 μg AR, 0.1 μg ARΔE/F] using Lipofectamine reagent (Gibco–BRL). Luciferase activity was determined with the Luciferase assay system (Promega). As a reference plasmid to normalize transfection efficiency, 2.5 ng pRL-CMV plasmid (Promega) was co-transfected. All values represent averages ±SD of at least three independent experiments.
In vitro ubiquitination assay. The in vitro ubiquitination assay was performed as previously described, with several modifications [16]. The CA-AhR immunocomplex was purified using anti-FLAG antibody from MCF-7 cells transfected with FLAG-HA-CA-AhR together with HA-DDB1 and myc-TBL3. The immunocomplex was incubated with recombinant ubiquitin and reaction buffer [50 mM Tris–HCl (pH 7.4), 5 mM MgCl, 2 mM NaF, 2 mM ATP and ATP-regenerating system, 0.6 mM DTT, and 12 μg ubiquitin (Calbiochem), 60 ng E1(Calbiochem), 0.3 μg E2 mixture set (Calbiochem)]. The self-ubiquitination of CA-AhR was detected by Western blotting using an anti-HA antibody.