α flag eomes m2

293T cells were transfected with wild-type or mutant T-bet and Eomes plasmids described above. After 48 hr, lysates were prepared in RIPA buffer and quantified using a Qubit (Thermo Fisher). Using the TransAM T-bet DNA binding ELISA kit (Active Motif), DNA binding of T-bet and Eomes was measured using a modified version of the manufacturers protocol (Figures S2B and S3A). For competition studies, 10 μg of lysate containing one transcription factor was allowed to bind to the plate for 1 hr at room temperature. Wells were then washed three times with wash buffer and 10 μg of lysate containing the other transcription factor was added to individual wells and allowed to bind for 1 hr at room temperature. Wells were seeded in duplicate. Recombinant T-bet and Eomes binding was detected using α–myc (T-bet) (9B10, Cell Signaling Technologies) or α–FLAG (Eomes) (M2, Sigma) monoclonal antibodies, respectively. For oligo competition studies, 40 pmol of competing oligos were added following addition of lysate to the wells. Protein binding was measured at OD₄₅₀ nm on a BioTek Synergy HT plate reader. Results are plotted as fold change from the uncompeted lysate containing WT T-bet or Eomes. Oligonucleotide sequences used in competition studies are listed in Table S2.