2.7. Western Blotting

MCF-7 cells were seeded in six-well plates at a density of 2 × 10⁵ cells per well. Forty-eight hours after serum starvation, cells were treated with test ligands for 4 or 24 h. Cells were lysed in RIPA buffer supplemented with 1X PIC and sonicated at a low intensity for 2 × 30 s on/off. The protein concentration of the clarified lysate was determined using the BCA assay according to the manufacturer’s protocol (Thermo Fisher Scientific). 40 µg of protein was separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. COS-1 cells were transfected with 1 µg of pEGFP-PARP7, pEGFP-PARP7 33–657, pEGFP-PARP7 53–657, pEGFP-PARP7 103–657, pEGFP-PARP7 200–657, pEGFP-PARP7 235–657, or pEGFP-PARP7 1–234. MEFs were seeded in six-well plates at a density of 1.0 × 10⁵ cells per mL. MCF-7 PARP7-HA cells were seeded in six-well plates at a density of 1.2 × 10⁵ cells per mL. After 24 h, these cells were incubated with and without 1.5 μg/mL DOX. The following day, cells were harvested and lysed in lysis buffer (200 mM NaCl, 20 mM Hepes, 1% Nonidet P-40), 20 µg of protein was separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with lab-generated anti-PARP7, anti-PARP7 (Abcam, Cambridge, UK; ab84664; lot# GR3304056-5), anti-ERα (MCF-7 cells only; Santa Cruz Biotechnology; HC-20), anti-GFP (COS-1 only; Clontech Laboratories, Mountain View, CA, USA; JL-8, anti-HA (MCF-7 PARP7-HA cells only; BioLegend, San Diego, CA, USA; 16B12) and anti-β-actin (Sigma-Aldrich; AC-74). After incubation with appropriate secondary antibody, bands were visualized with SuperSignal^TM West Dura Extended Duration Substrate or SuperSignal^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).