Odyssey emsa buffer kit

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).

MYC and MAX were translated individually or together in vitro using the TnT SP6 coupled wheat germ extract system (Promega), according to the manufacturer’s protocol. Plasmids used for MYC and MAX were pCS2-FLAG-hMYC and pRK7-HA-hMAX, respectively, and were generously provided by the Eisenman Lab (Fred Hutchinson Cancer Research Center). The protein concentrations of the in vitro translated products were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Binding reactions were carried out using Odyssey EMSA Buffer Kit (LI-COR), where 90–100 µg of the translated proteins were incubated with 7.5 nM IRDdye 700-labeled FGF7 WT or mutant DNA probes (IDT) in the presence or absence of their respective unlabeled competitor oligos (IDT), according to the manufacturer’s protocol. To separate the DNA–protein complex, the binding reactions were subjected to electrophoresis on a 6% DNA retardation gel (Thermo Fisher Scientific), which was then scanned using the Odyssey Infrared Imaging System (LI-COR) to detect the fluorescence signal. The assay was performed three times and showed similar results.