Monoclonal α myc antibody

Exogenous expression of recombinant myc-tagged T-bet or FLAG-tagged Eomes in 293T cells was determined by immunoblotting. Briefly, transfected 293T cells were pelleted and washed with 1x PBS. Pellets were resuspending in RIPA buffer (Sigma Aldrich) supplemented with protease inhibitor cocktail (Sigma Aldrich) and incubated on ice for 30 min. Lysates were then cleared by centrifugation for 10 min at 4°C. Lysates were quantified using a Qubit protein assay kit (Thermo Fisher). 25 μg of total lysate was resolved on a 4%–12% NuPage Bis Tris gel (Invitrogen) and transferred to a nitrocellulose membrane with an iBlot (Invitrogen). The nitrocellulose membrane was then blocked in 5% milk (Lab Scientific) in TBST for 1 hr at RT. Primary antibodies were added to 5% milk in TBST and rocked overnight at 4°C. T-bet was detected using a 1:1000 dilution of a monoclonal α-myc antibody (Cell Signaling). Eomes was detected using a 1:1000 dilution of a monoclonal α-FLAG antibody. As a loading control, a polyclonal α-α-actin antibody was used at a 1:2000 dilution. Blots were then washed 3x for 15 min in TBST and secondary α-mouse or α-rabbit HRP antibodies were added to 5% milk in TBST and rocked for 1hr at RT. Blots were then washed 3x for 15 min in TBST and proteins were detected using the Ultra ECL detection reagent (KwikQuant).