Western Blotting and Luciferase Experiments

NP-40 cell extracts were prepared and separated by denaturing SDS-PAGE as described (Saul et al., 2019). Proteins from SDS gels were transferred to polyvinylidene fluoride (PVDF) membranes using a semi-dry blotting apparatus (Bio-Rad) and 1 x transfer buffer [50 mM Tris; 40 mM glycine; 20% (v/v) methanol; 0.04% (w/v) SDS]. The electrophoretic transfer was performed at 24 V for 1–3 h, depending on the size of the proteins. After completion of protein transfer, the membranes were incubated in blocking buffer {5% (w/v) skimmed milk powder in TBS-T [25 mM Tris (pH 7.4); 137 mM NaCl; 5 mM KCl; 0.7 mM CaCl₂; 0.1 mM MgCl₂; 0.1% (v/v) Tween 20]}at room temperature for 1 h. The membranes were then incubated with the primary antibody solution at 4°C over night. Membranes were washed 5 × in TBS-T, followed by incubation with the secondary antibody solution for 1.5–2 h. The membrane was then washed again 5 times in TBS-T and proteins were detected on a ChemiDoc Touch imaging system (Bio-Rad) using enhanced chemiluminescence (ECL). Quantitative analysis of Western blot data was done in ImageLab 6.0.1 (Bio-Rad). Luciferase assays using the 293T-Gal4 cells were performed by transfection of the plasmids encoding the various Gal4 fusion proteins together with 100 ng of the plasmid encoding Renilla luciferase under the control of a constitutive SV40 promoter. After 2 days, cells were lysed in NP-40 lysis buffer as described (Saul et al., 2019) and extracts were either used for Western blotting or for determination of luciferase activities using the Dual Luciferase Assay System (Promega). Firefly luciferase activity was measured by mixing 2 μl of the extract with 10 μl of LAR II reagent, followed by determination of light emission using a Lumat LB9507 (Berthold Technologies). Renilla activity was measured in the same tube by adding 10 μl of Stop&Glo reagent, followed by determination of emitted light. Luciferase assays measuring ER-inducible gene expression were performed by transfection of cells with plasmids for lacO-(ERE)3-luciferase and SV40 Renilla luciferase together with plasmids encoding GFP-LacI-HIPK2, followed by further cultivation of cells in CSS-DMEM for 1 day. Then cells were treated either with DMSO as a vehicle control or with 17β-Estradiol (10 nM) for 8 h, followed by cell lysis in passive lysis buffer and determination of Renilla and Firefly luciferase activities. In all luciferase experiments, Firefly luciferase activities were normalized to the Renilla luciferase activities.