2.11. Co-Immunoprecipitation

COS-1 cells were seeded in six-well plates at a density of 2 × 10⁵ cells per well. The following day, cells were transfected with 0.5 µg of pCMV-FLAG-ERα, and either 1 µg pEGFP-PARP7, 0.8 µg pEGFP-PARP7^H532A or 0.1 µg of pEGFP using Lipofectamine 2000. Various amounts of pcDNA3.1 was used to reach a total amount of 1.5 µg DNA. Cells were treated with E2 for 24 h. For the truncated ERα variants, cells were transfected with 0.5 µg of pCMV-3xFLAG-ERα ABC, pCMV-3xFLAG-ERα ABCD, or pCMV-3xFLAG-ERα CDEF, and 1 µg of pEGFP-PARP7. The following day, cells were harvested and lysed in lysis buffer (200 mM NaCl, 20 mM Hepes, 1% Nonidet P-40). 10% of the lysate was kept as an input control, and the remaining lysate was incubated with 2 µg of anti-FLAG (Sigma-Aldrich; M2) and 20 µL of Dynabeads^TM Protein G (Thermo Fisher Scientific) with constant rotation for 2 h at 4 °C. The beads were washed five times with wash buffer (200 mM NaCl, 20 mM Hepes, 0.1% Nonidet P-40) and finally eluted in 2× Laemmli sample buffer supplemented with 10% β-mercaptoethanol. Samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with anti-poly/mono-ADP-ribose (Cell Signaling Technology, Danvers, MA, USA; E6F6A), anti-FLAG (Sigma-Aldrich; F7425), and anti-GFP (Clontech Laboratories; JL-8). The 10% totals were incubated with anti-FLAG, anti-GFP and anti-β-actin. After incubation with appropriate secondary antibody, bands were visualized with SuperSignal^TM West Dura Extended Duration Substrate (Thermo Fisher Scientific). Quantification of modified FLAG-ERα was done in ImageLab^TM (BioRad) by normalizing to β-actin of the respective samples.