Western Blotting of Ref-1 in TAIL7 Cells

Cell lysates were prepared in RIPA lysis buffer system (Santa Cruz Biotechnology, Dallas, TX), as described (21 (link), 22 (link)). All experiments with TAIL7 cells were performed using IL-7 (10ng/ml). For studies of Ref-1 regulation by glucocorticoids, TAIL7 cells were incubated with Dexamethasone for the timepoints indicated. Equal amounts of protein (20–50mg/sample) were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with antibodies for Ref-1 (Novus Biologicals, Littleton, CO), or for Actin (Thermo Fisher Scientific, Waltham, MA) as loading control. Immunodetection was performed by incubation with HRP-conjugated anti-mouse IgG antibodies (EMD Millipore, Billerica, MA), followed by chemiluminescence developing using WesternBright Quantum Western blotting detection kit (Advansta, Menlo Park, CA). Determination of relative protein intensity was performed using Quantity One software (Bio-Rad, Hercules, CA).