Protein-DNA Binding Assay in Trophoblast Cells

Immortalized extravillous trophoblast cell line HTR8/SVneo (obtained from the American Type Culture Collection, item number CRL-3271) was transfected with Lipofectamine3000 (ThermoFisher Scientific) and overexpression vector (DLX3 or GATA2 Myc-DDK vectors, Origene) for 72 hours according to manufacturer protocol. Cytoplasmic protein fraction was isolated by suspending cell pellet in cytoplasmic lysis buffer (10 mM HEPES, 10 mM KCl, 1.1 mM EDTA in dH₂O) and adding 10% Nonidet-P40. Nuclear protein fraction was subsequently isolated by suspending nuclear pellet in nuclear lysis buffer (20 mM HEPES, 0.4 M KCl, 1 mM EDTA in dH₂O). DNA probes were generated with IRDye700 at the 5′ end (GATA2 TCR probe: 5′-CACTTGATAACAGAAAGTGATAACTCT-3′ [78 (link)], DLX3 JRE probe: 5′-GGGGGGTAATTACAGGCCC-3′ [79 (link)], THE1B 5′ probe: 5′-GGATAATGATATGGTTAGA-3′). Binding reactions were performed using the Odyssey EMSA Buffer Kit (LICOR Biosciences) according to manufacturer protocol with primary antibody (monoclonal anti-FLAG M2, Sigma-Aldrich F3165) or isotype control (mouse IgG, Abcam ab37355) and run on 6% TBE gels with 0.5× TBE buffer (ThermoFisher Scientific). Gels were imaged on the Odyssey CLx Imaging System (LICOR Biosciences).