Immobilon western chemiluminescence horseradish peroxidase substrate

Immune cells or tumour cells treated with 2.5, 5, 7.5 and 15 μM of L971 with or without cytokine treatment: 20 ng/ml IL6, 50 ng/ml IFNβ and 20 ng/ml TNFα for 10 minutes, or 100 ng/ml LPS for 0.5 hours. Cells were washed twice with cold PBS and harvested in lysis buffer containing protease and phosphatase inhibitors. Total of 20 μg protein lysates were resolved by SDS‐PAGE electrophoresis gel and transferred onto nitrocellulose membranes (GE Healthcare, Cat. 10600034). After blocking with nonfat milk solution (with 0.5‰ Tween‐20), the membranes were probed with primary antibodies at 4℃ overnight and then incubated with horseradish peroxidase‐conjugated secondary antibodies for 2 hours at room temperature. Immune complexes were detected with an Immobilon™ western chemiluminescence horseradish peroxidase substrate (Millipore, Cat. WBKLS0500) and photographed with a Tanon 5200 imaging system. The quantification was managed by ImageJ software.

The recombinant wild type PTEN and Trx1 were purified as previously described [27 (link)]. Thioredoxin reductase was purified from the mouse liver as described previously [49 (link)]. β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), N-ethylmaleimide (NEM), N,N-dimethylformamide (DMF), and dl-dithiothreitol (dl-DTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide (H₂O₂) was purchased from OCI Company Ltd. (Seoul, Korea). tert-Butyl hydroperoxide (t-BHP) was purchased from Alfa Aesar (Ward Hill, MA, USA). Protease inhibitor (Complete ULTRA Tablets) was purchased from Roche Diagnostics GmbH (Indianapolis, IN, USA). PTEN antibody was prepared as previously described [50 (link)]. Anti-Trx1 and anti-rabbit IgG horseradish peroxidase-conjugated antibodies were purchased from Ab Frontier (Daejeon, Korea). Anti-tubulin antibodies were from Sigma-Aldrich. Immobilon Western chemiluminescence horseradish peroxidase substrate was purchased from Millipore Corporation (Billerica, MA, USA). NAP-5 Columns (Sephadex G-25 DNA Grade) were purchased from GE Healthcare (Little Chalfont, UK). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany).