FIG. 3.

TReP-132 interacts directly with progesterone-bound PR via its LXXLL NR-boxes. A. Coimmunoprecipitation assays. T47D cells were treated or not with progesterone (30 nM) and RU486 (10 nM) for 12 h. Total protein extracts (30 μg) were then submitted to Western blot analyses using a PR antibody either after immunoprecipitation with anti-TReP-132 or nonimmune rabbit IgG (negative control) antibodies (upper panel) or directly for control of the in-cell PR levels (lower panel). B. GST pull-down assays. Top panel: GST-TReP-132 immobilized on glutathione-coupled Sepharose was incubated with [³⁵S]methionine-labeled PR (−) with increasing concentrations of progesterone (0 to 100 μM; left panel) or with 100 μM progesterone together with increasing doses of RU486 (0 to 100 μM; right panel). Specificity of interaction was assessed by comparison with background levels obtained by incubating GST alone with [³⁵S]methionine-labeled PR in the presence of 100 μM progesterone (GST-control). Lower panel: GST-TReP-132, either wild type or mutated in the LRQLL (GST-TReP-132m1) or LEMLL (GST-TReP-132m2) motifs, was assayed for interaction with [³⁵S]PR as described above in the presence of progesterone (10 μM). PR input contains 1/10 the amount of radioactive proteins used in the incubations.