Westernbright quantum western blotting detection kit

Tumors were homogenized in lysis buffer (10 mmol l⁻¹ Tris-HCl pH 7.5, 150 mmol l⁻¹ sodium chloride, 10 mmol l⁻¹ sodium pyrophosphate, 1 mmol l⁻¹ β-glycerolphosphate, 1 mmol l⁻¹ sodium orthovanadate, 50 mmol l⁻¹ sodium fluoride, 1.25% v/v CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and Complete® Protease Inhibitor Cocktail (Roche Diagnostics). Lysates were rotated at 4°C for 1 h and centrifuged at 13,000 rpm for 10 min. Supernatants were collected and protein concentrations were determined with Protein Assay Kit (Bio-Rad, Richmond, California). Protein (12 μg) was electrophoresed through a 10% polyacrilamide gel and transferred to a nitrocellulose membrane. The membranes were immunoblotted with the desired antibody, followed by an appropriate secondary antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania). Immunoreactivity was detected by enhanced chemiluminescence (WesternBright™ Quantum Western blotting detection kit, Advansta, Melano Park, California), using luminescent image analyzer LAS-4000 (Fujifilm, Tokyo, Japan). Densitometry analysis was performed using ImageQuant software (GE Healthcare Bio-Sciences, Pittsburgh, Pennsylvania).
The following antibodies were used: Phospho-Akt (Thr308), total-Akt, and β-actin (Cell Signaling Technology, Danvers, Massachusetts).

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).